Enhanced erythropoiesis and iron metabolism

ABSTRACT

The present invention relates to methods for treating anemia in a subject having a low percent transferrin saturation by administering to the subject an effective amount of a compound that inhibits hypoxia-inducible factor prolyl hydroxylase activity.

This application is a continuation of U.S. application Ser. No.10/861,590, filed on 3 Jun. 2004, which claims the benefit of U.S.Provisional Application Ser. No. 60/476,704, filed on 6 Jun. 2003; U.S.Provisional Application Ser. No. 60/566,488, filed on 29 Apr. 2004; U.S.Provisional Application Ser. No. 60/566,237, filed on 29 Apr. 2004; andU.S. Provisional Application Ser. No. 60/569,797, filed on 10 May 2004,each of which is incorporated by reference herein in its entirety.

FIELD OF THE INVENTION

The present invention relates to methods and compounds for regulating orenhancing erythropoiesis and iron metabolism, and for treating orpreventing iron deficiency and anemia of chronic disease.

BACKGROUND OF THE INVENTION

Anemia generally refers to any abnormality in hemoglobin or erythrocytesthat leads to reduced oxygen levels in the blood. Anemia can alsodevelop in association with chronic diseases, e.g., chronic infection,neoplastic disorders, chronic inflammatory disorders, includingdisorders with consequent inflammatory suppression of marrow, etc.Anemia of chronic disease is one of the most common syndromes inmedicine.

Anemia of chronic disease (ACD) is often associated with irondeficiencies. ACD can develop from inadequate availability of iron(e.g., anemia of iron deficiency) or, in cases where total body iron isadequate but the requirements for hemoglobin production are defective(e.g., functional iron deficiency). Iron is required for production ofred blood cell hemoglobin in erythropoietic precursor cells of the bonemarrow.

Numerous physiologic deficiencies are observed in patients with anemiaof chronic disease, including reduced erythropoietin (EPO) production,reduced EPO responsiveness of the bone marrow, and reduced ironmetabolism, including reduced iron absorption from the gut, reduced irontrans-enterocyte transport, reduced iron oxidation to the ferric stateby hephaestin or ceruloplasmin, reduced iron binding and uptake bytransferrin and transferrin receptor, and reduced iron transport to themarrow where iron utilization occurs, including heme synthesis.Individually and together, these physiologic deficiencies contribute toineffective or impaired erythropoiesis, which can lead to microcyticanemia and hypochromic red blood cells associated with reducedhemoglobin production and reduced oxygen transport.

Anemia of chronic disease is associated with increased production ofinflammatory cytokines (Means (1995) Stem cells 13:32-37 and Means(1999) Int J Hematol 70:7-12), including, for example, tumor necrosisfactor-α (TNF-α), interleukin-1β (IL-1β), IL-6, and interferon-γ(IFN-γ). In several in vitro and in vivo animal model systems,inflammatory cytokines negatively affected the ability to mediate EPOproduction, EPO responsiveness, and the coordinate regulation of ironmetabolism (Roodman et al. (1989) Adv Exp Med Biol 271:185-196; Fuchs etal. (1991) Eur J Hematol 46:65-70; Jelkmann et al. (1994) Ann NY AcadSci 718:300-311; Vannucchi et al. (1994) Br J Hematol 87:18-23; andOldenburg et al. (2001) Aliment Pharmacol Ther 15:429-438.)Administration of erythropoietin failed to reverse anemia in micecontinuously exposed to TNF-α (Clibon et al. (1990) Exp Hematol18:438-441). Increased levels of inflammatory cytokines, such as TNF-α,IL-1β, and INF-γ, contribute to defective EPO production and EPOresistance observed in patients with anemia of chronic disease (Jelkmannet al. (1991) Ann NY Acad Sci 718:300-311 and Macdougall and Cooper(2002) Neprol Dial Transplant 17(11):39-43). Therefore, variouscytokines, e.g., inflammatory cytokines and cytokines associated withinflammation, are involved in many aspects of the pathogenesis of anemiaof chronic disease, including inhibition of erythroid progenitors,inhibition of EPO production, and impairment of iron release and ironavailability for erythropoiesis.

There is thus a need in the art for methods of treating or preventinganemia of chronic disease. There is a need in the art for methods ofovercoming the deficiencies in current use of recombinant EPO to treatanemia of chronic disease. In particular, there remains a need formethods and compounds effective at overcoming suppressed EPO productionand decreased EPO responsiveness associated with anemia of chronicdisease, for methods and compounds effective at enhancing regulation ofiron metabolism and overcoming deficiencies of altered or abnormal ironmetabolism and utilization, and for methods and compounds effective atenhancing total or complete erythropoiesis by improving the metabolicpathways related to EPO production, EPO responsiveness and signaling,and iron availability, utilization, uptake, transport, processing, etc.There is a need in the art for methods of overcoming or of amelioratingthe consequences of cytokine-induced effects in subjects having anemiaof chronic disease.

Iron deficiency is one of the most common nutritional deficienciesworldwide and is the leading cause of anemia on a global basis. Ironbalance is fundamentally regulated by the rate of erythropoiesis and thesize of iron stores. Iron deficiency can occur with or without anemia,and has been associated with impaired cognitive development.

Iron deficiency is defined as inadequate iron supply (levels or stores)or as inadequate availability or utilization of iron in the body. Thiscan be due to nutritional deficiencies, e.g., lack of iron in the diet;to iron malabsorption, due, for example, to surgery (post-gastrectomy)or disease (Crohn's disease); or to a depletion in iron supply orincreased iron loss due to chronic or acute blood loss resulting frominjury or trauma, menses, blood donation, phlebotomy (such as due tovarious procedures, surgeries); from increased iron demand, e.g., due torapid growth in infancy or adolescence, pregnancy, erythropoietintherapy, etc.

Iron deficiency can also be functional iron deficiency, e.g., irondeficiency characterized by the subject's impaired ability to access andutilize iron stores. Iron is not available at a rate sufficient to allownormal hemoglobinization of erythrocytes, leading to reducedreticulocyte and erythrocyte cellular hemoglobin content. Functionaliron deficiency is often seen in healthy individuals with apparentlynormal or even increased iron stores but with impaired ironavailability, as measured, e.g., by low levels of percent transferrinsaturation. This type of iron deficiency is frequently associated withacute or with chronic inflammation.

Iron deficiency of any kind can lead to iron-deficient oriron-restricted erythropoiesis, in which red blood cell numbers decreaseand circulating red blood cells are smaller than normal (microcytic) andlack adequate hemoglobin, and as such are pale in color (hypochromic).

Subjects with iron deficiency, including functional iron deficiency, candevelop impaired hemoglobin synthesis, reduced % transferrin saturation,and decreased hemoglobin and hematocrit levels, leading to irondeficiency anemia. Iron deficiency anemia is the most common anemia inthe world. Iron is an essential component of hemoglobin; without iron,the marrow is unable to produce hemoglobin effectively. Iron deficiencyanemia may occur in subjects with depleted or impaired iron supply, ormay occur in subjects having functional iron deficiency, when iron ispresent in storage but is unavailable, e.g., for hemoglobin production.

In view of the above, there is a need in the art for methods of treatingor preventing disorders associated with iron metabolism, and a need inthe art for methods of enhancing iron metabolism. There is a need formethods of treating or preventing iron deficiency, including functionaliron deficiency, and for treating or preventing associated conditionssuch as microcytosis and iron deficiency anemia.

The present invention provides methods and compounds for enhancing themetabolic and physiologic pathways that contribute to complete andeffective erythropoiesis, and in particular, for treating anemia ofchronic disease. Methods and compounds for overcoming thesuppressive/inhibitory effects of inflammatory cytokines on EPOproduction and responsiveness are also provided. Additionally thepresent invention provides methods and compounds for enhancing ironmetabolism, and for treating or preventing conditions associated withimpaired iron metabolism, such as iron deficiency, including functionaliron deficiency, iron deficiency anemia, microcytosis, iron-deficienterythropoiesis, etc.

SUMMARY OF THE INVENTION

The present invention relates to methods and compounds for inducingenhanced or complete erythropoiesis in a subject. In particular, themethods comprise inducing enhanced or complete erythropoiesis bystabilizing HIFα in a subject. Methods of inducing enhancederythropoiesis by inhibiting HIF prolyl hydroxylase are specificallycontemplated. In specific embodiments, the methods compriseadministering to a subject a compound of the invention. In variousembodiments, the subject can be a cell, tissue, organ, organ system, orwhole organism.

The subject is, in various embodiments, a cell, tissue, organ, organsystem, or whole organism. In particular embodiments, the organism is amammal, preferably, a human.

In one aspect, the method increases the production of factors requiredfor differentiation of erythrocytes from hematopoietic progenitor cellsincluding, e.g., hematopoietic stem cells (HSCs), CFU-GEMM(colony-forming-unit-granulocyte/erythroid/monocyte/megakaryocyte)cells, etc. Factors that stimulate erythropoiesis include, but are notlimited to, erythropoietin. In another aspect, the methods increase theproduction of factors required for iron uptake, transport, andutilization. Such factors include, but are not limited to, erythroidaminolevulinate synthase, transferrin, transferrin receptor,ceruloplasmin, etc. In yet another aspect, the method increases factorsrequired for differentiation of erythrocytes and additionally factorsrequired for iron uptake, transport, and utilization.

In another embodiment, the methods of the invention enhanceresponsiveness of hematopoietic precursors to erythropoietin. Asdescribed above, such precursors include HSCs, CFU-GEMMs, etc. Theresponsiveness of the precursor cells can be augmented, e.g., byaltering expression of erythropoietin receptors, intracellular factorsinvolved in erythropoietin signaling, and secreted factors thatfacilitate interaction of erythropoietin with the receptors.

In another aspect, the methods can be used to overcome inhibition oferythropoiesis induced by inflammatory cytokines such as tumor necrosisfactor-α (TNF-α), interleukin-1β (IL-1β), and the like. In particularaspects, the methods can be used to treat anemia that is refractive totreatment with exogenously administered erythropoietin. Such anemia canbe caused, e.g., by chronic inflammatory or autoimmune disordersincluding, but not limited to, chronic bacterial endocarditis,osteomyelitis, rheumatoid arthritis, rheumatic fever, Crohn's disease,and ulcerative colitis.

In certain embodiments, the methods of the invention can be used totreat anemia of chronic disease. Methods for inducing enhanced orcomplete erythropoiesis in patients with anemia of chronic disease arespecifically provided. In particular embodiments, the methods increasethe amount of iron available to make new red blood cells.

In another aspect, the present invention provides methods for enhancingEPO responsiveness of the bone marrow.

Methods for inhibiting TNFα suppression of EPO are specificallyprovided, as are methods for inhibiting IL-1β suppression of EPO.

The present invention relates to methods for the treatment/prevention ofanemia of chronic disease, and methods for regulation of iron processingand treatment/prevention of conditions associated with deficiencies iniron and/or iron processing.

In one aspect, the invention provides a method for treating anemia ofchronic disease in a subject, the method comprising administering to thesubject an effective amount of a compound that stabilizes the alphasubunit of hypoxia inducible factor (HIF), thereby treating anemia ofchronic disease in the subject. Methods for achieving specificphysiological effects in a subject having anemia of chronic disease arealso provided; in particular, methods for increasing reticulocytes,increasing mean corpuscular cell volume, increasing mean corpuscularhemoglobin, increasing hematocrit, increasing hemoglobin, and increasingred blood cell count, etc., in a subject having anemia of chronicdisease, each method comprising administering to the subject aneffective amount of a compound that stabilizes the alpha subunit ofhypoxia inducible factor (HIF), thereby achieving the desiredphysiological effect. In various aspects, the anemia of chronic diseaseis associated with, e.g., inflammation, autoimmune disease, irondeficiency, microcytosis, malignancy, etc.

In various embodiments, the subject is a cell, tissue, or organ. Inother embodiments, the subject is an animal, preferably a mammal, mostpreferably a human. When the subject is a cell, the inventionspecifically contemplates that the cell can be an isolated cell, eitherprokaryotic or eukaryotic. In the case that the subject is a tissue, theinvention specifically contemplates both endogenous tissues and in vitrotissues, e.g., tissues grown in culture. In preferred embodiments, thesubject is an animal, particularly, an animal of mammalian speciesincluding rat, rabbit, bovine, ovine, porcine, murine, equine, andprimate species. In a most preferred embodiment, the subject is human.

Stabilization of HIFα can be accomplished by any of the methodsavailable to and known by those of skill in the art, and can involve useof any agent that interacts with, binds to, or modifies HIFα or factorsthat interact with HIFα, including, e.g., enzymes for which HIFα is asubstrate. In certain aspects, the present invention contemplatesproviding a constitutively stable HIFα variant, e.g., stable HIEmuteins, etc, or a polynucleotide encoding such a variant. In otheraspects, the present invention contemplates that stabilizing HIFαcomprises administering an agent that stabilizes HIFα. The agent can becomposed of polynucleotides, e.g. antisense sequences; polypeptides;antibodies; other proteins; carbohydrates; fats; lipids; and organic andinorganic substances, e.g., small molecules, etc. In a preferredembodiment, the present invention contemplates stabilizing HIFα, e.g.,in a subject, by administering to the subject an agent that stabilizesHIFα wherein the agent is a compound, e.g., small molecule compound,etc., that stabilizes HIFα.

In various aspects, HIFα is HIF1α, HIF2α, or HIF3α. In a preferredaspect, stabilizing HIFα comprises administering to the subject aneffective amount of a compound that inhibits HIF hydroxylase activity.In certain aspects, the HIF hydroxylase is selected from the groupconsisting of EGLN1, EGLN2, and EGLN3.

In one embodiment, the invention provides a method for increasing meancorpuscular volume in a subject, the method comprising administering tothe subject an effective amount of a compound that stabilizes the alphasubunit of hypoxia inducible factor (HIF). In a further embodiment, theinvention provides a method for increasing mean corpuscular hemoglobinlevels in a subject, the method comprising administering to the subjectan effective amount of a compound that stabilizes the alpha subunit ofhypoxia inducible factor (HIF). In another embodiment, the presentinvention encompasses a method for reducing microcytosis in a subject,the method comprising administering to the subject an effective amountof a compound that stabilizes the alpha subunit of hypoxia induciblefactor (HIF).

The invention further provides a method for treating or preventingmicrocytic anemia, the method comprising administering to the subject aneffective amount of a compound that stabilizes the alpha subunit ofhypoxia inducible factor (HIF).

In one aspect, the invention relates to a method for treating orpreventing a condition associated with iron deficiency in a subject, themethod comprising administering to the subject an effective amount of acompound that stabilizes the alpha subunit of hypoxia inducible factor(HIF). In a particular aspect, the invention provides a method forimproving iron processing in a subject, the method comprisingadministering to the subject an effective amount of a compound thatstabilizes the alpha subunit of hypoxia inducible factor (HIF). A methodfor treating or preventing a condition associated with compromised ironavailability in a subject is also provided, the method comprisingadministering to the subject an effective amount of a compound thatstabilizes the alpha subunit of hypoxia inducible factor (HIF).

In other embodiments, the invention relates to a method for overcomingcytokine-induced effects in a subject. In particular, the inventionprovides in one aspect a method for overcoming cytokine-suppression ofEPO production in a subject, the method comprising administering to thesubject an effective amount of a compound that stabilizes the alphasubunit of hypoxia inducible factor (HIF). The invention furtherprovides a method for overcoming cytokine-suppression of ironavailability in a subject, the method comprising administering to thesubject an effective amount of a compound that stabilizes the alphasubunit of hypoxia inducible factor (HIF). In another aspect, thepresent invention encompasses a method for treating or preventingcytokine-associated anemia in a subject, the method comprisingadministering to the subject an effective amount of a compound thatstabilizes the alpha subunit of hypoxia inducible factor (HIF). Methodsfor increasing EPO production in the presence of a cytokine in asubject, the methods comprising administering to the subject aneffective amount of a compound that stabilizes the alpha subunit ofhypoxia inducible factor (HIF), are also provided. In specificembodiments, the cytokine is selected from the group consisting of TNF-αand IL-1β.

In one aspect, the invention provides a method for reducingcytokine-induced VCAM expression in a subject, the method comprisingadministering to the subject an effective amount of a compound thatstabilizes the alpha subunit of hypoxia inducible factor (HIF). In aspecific aspect, the cytokine is TNF-α or IL-1β. In one aspect, themethod applies to reduction of cytokine-induced VCAM expression inendothelial cells in the subject. In another aspect, the subject has acondition selected from the group consisting of inflammatory disease,autoimmune disease, and anemia of chronic disease.

In another aspect, the invention provides a method for reducingcytokine-induced E-selectin expression in a subject, the methodcomprising administering to the subject an effective amount of acompound that stabilizes the alpha subunit of hypoxia inducible factor.In a specific aspect, the cytokine is TNF-α or IL-1β. In one aspect, themethod applies to reduction of cytokine induced E-selectin expression inendothelial cells in the subject. In another aspect, the subject has acondition selected from the group consisting of inflammatory disease,autoimmune disease, and anemia of chronic disease.

The invention provides various methods of regulating/enhancing ironprocessing and iron metabolism. In one aspect, the invention providesmethods for increasing iron transport, uptake, utilization, andabsorption in a subject, each of the methods comprising administering tothe subject an effective amount of a compound that stabilizes the alphasubunit of hypoxia inducible factor (HIF). In particular embodiments,the invention provides methods for increasing transferrin expression,transferrin receptor expression, IRP-2 expression, ferritin expression,ceruloplasmin expression, NRAMP2 expression, sproutin expression, andALAS-2 expression in a subject, each method comprising administering tothe subject an effective amount of a compound that stabilizes the alphasubunit of hypoxia inducible factor (HIF). In other embodiments, theinvention provides methods for decreasing hepcidin expression, themethod comprising administering to the subject an effective amount of acompound that stabilizes the alpha subunit of hypoxia inducible factor(HIF). Methods for increasing heme synthesis in a subject byadministering to the subject an effective amount of a compound thatstabilizes the alpha subunit of hypoxia inducible factor (HIF) are alsoprovided.

In certain aspects, the invention contemplates methods for increasingserum iron, increasing transferrin saturation, increasing solubletransferrin receptor levels, and increasing serum ferritin levels in asubject, the methods comprising administering to the subject aneffective amount of a compound that stabilizes the alpha subunit ofhypoxia inducible factor (HIF). In a further aspect, the inventionprovides a method for increasing iron transport to bone marrow in asubject, the method comprising administering to the subject an effectiveamount of a compound that stabilizes the alpha subunit of hypoxiainducible factor (HIF).

In one aspect, the present methods are applied to treatment of ormanufacture of a medicament for a subject, preferably a human subject,having any of the disorders and conditions discussed herein. It is to beunderstood that various parameters associated with clinical conditionsvary according to age, gender, etc. In one aspect, the subject has aserum ferritin level below normal range, e.g., below 50-200 μg/L; thus,a subject having serum ferritin levels below 200 ng/ml, below 150 ng/ml,below 100 ng/ml, below 75 ng/ml, and below 50 ng/ml could be a suitablesubject for treatment with the methods or use of medicaments provided bythe present invention. Alternatively, a suitable subject could beidentified by demonstrating a total iron-binding capacity (TIBC) of lessthan normal range, e.g., less than TIBC 300-360 μg/dL.

In another embodiment, the subject has a serum iron level below thenormal range, e.g., below serum iron levels of 50-150 μg/dL. Otherappropriate parameters for identifying suitable subjects includetransferrin saturation measurements of below 30-50%, marrow sideroblastmeasurements of below 40-60%, and hemoglobin levels of below about 10 to11 g/dL. Any of the above parameters are measured, e.g., as in standardhematological tests, blood chemistry and complete blood count (CBC)analysis, typically presented as a measurement of several bloodparameters, and obtained, e.g., by analysis of blood by an automatedinstrument which measures, for example, red blood cell count, whiteblood cell count, platelet count, and red cell indices. Measurement maybe by any standard means of measurement of hematological and/orbiochemical blood analysis, including, e.g., automated systems such asthe CELL DYN 4000 analyzer (Abbott Laboratories, Abbott Park Ill.), theCoulter GenS analyzer (Beckman Coulter, Inc., Fullerton Calif.), theBayer ADVIA 120 analyzer (Bayer Healthcare AG, Leverkusen, Germany),etc.

In one aspect, the invention encompasses a method for treating orpreventing iron deficiency in a subject, the method comprisingadministering to the subject an effective amount of a compound thatstabilizes HIFα, thereby treating or preventing iron deficiency in thesubject. In further aspects, the iron deficiency is functional irondeficiency; is associated with anemia; is associated with a disorderselected from the group consisting of an inflammation, infection,immunodeficiency disorder, and neoplastic disorder; or is associatedwith a disorder selected from the group consisting of anemia of chronicdisease, iron deficiency anemia (IDA), and microcytic anemia.

A subject of the invention could be a subject with any clinicallyaccepted standard measurement indicative of iron deficiency or of a riskfor developing iron deficiency. For example, in certain embodiments, thesubject has low serum ferritin levels (<20 ng/ml), or reduced %transferrin saturation, e.g., less than 16% (in adults). Serum ferritinlevels of below 50 ng/ml, below 40 ng/ml, below 30 ng/ml, and below 20ng/ml are specifically contemplated. It is noted that if the subject hasor is at risk for having an iron deficiency that is functional irondeficiency, the serum ferritin levels could be increased above normalrange, e.g., 200 ng/ml and above. Iron deficiency can be observedthrough onset of iron-restricted/iron-deficient erythropoiesis(impairment of hemoglobin synthesis that is observed typically when %transferrin saturation falls below 15 to 20%). These iron parameters canbe measured using any standard CBC or biochemical analysis describedabove, and/or by use of automated devices more specifically directed toiron analysis, e.g., the Unimate 5 Iron and Unimate 7 UIBC kits (Roche,Switzerland).

A subject that might benefit from the present methods of treating orpreventing could be a subject having or at risk for having irondeficiency anemia; for example, a subject having a transferrinsaturation % of 10-15% or of below 10%.

In one aspect, the subject having or at risk for having iron deficiencyhas or is at risk for having functional iron deficiency. A reticulocytehemoglobin content of less than 28 picograms/cell could be indicative ofsuch a condition. In another aspect, the subject having or at risk forhaving functional iron deficiency displays greater than 5% hypochromicred cells.

In certain embodiments, the subject is one having or at risk for havinganemia of chronic disease. Such a subject could display mild or moderateanemia, e.g., hemoglobin levels of around 10-13 g/dL, or, moreparticularly, 10-11 g/dL. In other embodiments, more acute anemia isdisplayed, e.g., hemoglobin levels below 10 g/dL, including levels below5 g/dL, and levels below 3 g/dL. In some embodiments, the subject havingor at risk for having anemia of chronic disease displays abnormalitiesin iron distribution. Such abnormalities could be, e.g., serum ironlevels below around 60 μg/dL, or serum ferritin levels above normalrange, e.g., of above 200 ng/ml, above 300 ng/ml, or above 400 ng/ml.

In certain aspects, the subject could have or be at risk for havingmicrocytic anemia. Such a subject may, for example, demonstrate a meancorpuscular volume of less than 80 femtoliters measured, e.g., as partof complete blood count analysis. In other aspects, the subject has amean corpuscular volume of less than the normal value of 90+/−8femtoliters. The subject can have, in various aspects, a reduced meancell hemoglobin count, for example, a mean cell hemoglobin count of lessthan 30+/−3 picograms of hemoglobin/cell; or a reduced mean cellhemoglobin concentration, e.g., a mean cell hemoglobin concentration ofless than 33+/−2%.

A method for treating or preventing functional iron deficiency in asubject, the method comprising administering to the subject an effectiveamount of a compound that stabilizes HIFα, thereby treating orpreventing functional iron deficiency, is also provided.

In one embodiment, the present invention provides a method forregulating or enhancing iron metabolism or an iron metabolic process ina subject, the method comprising administering to the subject aneffective amount of a compound that stabilizes HIFα, thereby regulatingor enhancing iron metabolism or the iron metabolic process in thesubject. In another embodiment, the invention provides a method forregulating or enhancing an iron metabolic process selected from thegroup consisting of iron uptake, iron absorption, iron transport, ironstorage, iron processing, iron mobilization, and iron utilization, themethod comprising administering to the subject an effective amount of acompound that stabilizes HIFα, thereby regulating or enhancing the ironmetabolic process in the subject.

A method for increasing iron absorption in a subject, the methodcomprising administering to the subject an effective amount of acompound that stabilizes HIFα, thereby increasing iron absorption in thesubject, is also provided herein. In certain aspects, the ironabsorption is in the intestine; is absorption of dietary iron; or is induodenal enterocytes.

The following methods are also contemplated herein: a method forincreasing iron transport in a subject, the method comprisingadministering to the subject an effective amount of a compound thatstabilizes HIFα, thereby increasing iron transport in the subject; amethod for increasing iron storage in a subject, the method comprisingadministering to the subject an effective amount of a compound thatstabilizes HIFα, thereby increasing iron storage in the subject; amethod for increasing iron uptake in a subject, the method comprisingadministering to the subject an effective amount of a compound thatstabilizes HIFα, thereby increasing iron uptake in the subject; a methodfor increasing iron processing in a subject, the method comprisingadministering to the subject an effective amount of a compound thatstabilizes HIFα, thereby increasing iron processing in the subject; amethod for increasing iron mobilization in a subject, the methodcomprising administering to the subject an effective amount of acompound that stabilizes HIFα, thereby increasing iron mobilization inthe subject; and a method for increasing iron utilization in a subject,the method comprising administering to the subject an effective amountof a compound that stabilizes HIFα, thereby increasing iron utilizationin the subject.

In one embodiment, the invention contemplates a method for increasingiron availability for erythropoiesis in a subject, the method comprisingadministering to the subject an effective amount of a compound thatstabilizes HIFα, thereby increasing iron availability for erythropoiesisin the subject. In various embodiments, the increasing iron availabilityfor erythropoiesis is increasing iron availability for heme synthesis;is increasing iron availability for hemoglobin production; or isincreasing iron availability for red blood cell production.

The invention further provides methods for regulating expression of ironregulatory factors in a subject, the method comprising administering tothe subject an effective amount of a compound that stabilizes HIFα,thereby regulating expression of iron metabolic factors in the subject.

Methods for increasing expression of certain iron regulatory factors areencompassed herein, including: a method for increasing transferrinreceptor expression in a subject, the method comprising administering tothe subject an effective amount of a compound that stabilizes HIFα,thereby increasing transferrin receptor expression in the subject; amethod for increasing transferrin expression in a subject, the methodcomprising administering to the subject an effective amount of acompound that stabilizes HIFα, thereby increasing transferrin expressionin the subject; a method for increasing ceruloplasmin expression in asubject, the method comprising administering to the subject an effectiveamount of a compound that stabilizes HIFα, thereby increasingceruloplasmin expression in the subject; a method for increasing NRAMP2(slc11a2) expression in a subject, the method comprising administeringto the subject an effective amount of a compound that stabilizes HIFα,thereby increasing NRAMP2 expression in the subject; a method forincreasing duodenal cytochrome b reductase 1 expression in a subject,the method comprising administering to the subject an effective amountof a compound that stabilizes HIFα, thereby increasing duodenalcytochrome b reductase 1 expression in the subject; and a method forincreasing 5-aminolevulinate synthase expression in a subject, themethod comprising administering to the subject an effective amount of acompound that stabilizes HIFα, thereby increasing 5-aminolevulinatesynthase expression in the subject.

In one embodiment, the invention provides a method for increasing serumiron in a subject, the method comprising administering to the subject aneffective amount of a compound that stabilizes HIFα, thereby increasingserum iron in the subject. In certain embodiments, the subject is ahuman, and the serum iron levels are increased to a value between 50 to150 μg/dL.

In another aspect, the present invention provides methods for increasingtotal iron-binding capacity (TIBC) in a subject. The method comprisingadministering to the subject an effective amount of a compound thatstabilizes HIFα, thereby increasing TIBC in the subject. In a preferredaspect, the subject is a human and the total iron-binding capacity isincreased to a value between 300 to 360 μg/dL.

Methods and compounds for modulating serum ferritin levels in a subjectare provided. In a certain embodiment, the subject is a human, and theserum ferritin levels are increased above 15 μg/L. In a furtherembodiment, the subject is a human adult male, and the serum ferritinlevel is increased to a value of about 100 μg/L. In another embodiment,the subject is a human adult female, and the serum ferritin level isincreased to a level of about 30 μg/L.

In one aspect, the invention includes a method for increasingtransferrin saturation in a subject, the method comprising administeringto the subject an effective amount of a compound that stabilizes HIFα,thereby increasing transferrin saturation in the subject. In one aspect,the transferrin saturation is increased above a level selected from thegroup consisting of 10%, 15%, 20%, 30%, 40%, and 50%. The presentinvention encompasses methods for increasing percent transferrinsaturation in a subject. In one embodiment, the subject is a human andthe percent transferrin saturation is increased to a value above 18%. Inanother embodiment, the percent transferrin saturation is increased to avalue between 25 to 50%. Percent transferrin is typically calculatedusing the formula: (serum iron)(100)/(TIBC).

Methods for increasing soluble transferrin receptor levels in a subject,the methods comprising administering to the subject an effective amountof a compound that stabilizes HIFα, thereby increasing solubletransferrin receptor levels in the subject, are also provided. Theinvention further provides methods for increasing total erythroid marrowmass as measured by, e.g., serum transferrin receptor levels. In oneaspect, the subject is human and the serum transferrin receptor level isincreased to 4 to 9 μg/L as determined by immunoassay.

A method for decreasing hepcidin expression in a subject is provided,the method comprising administering to the subject an effective amountof a compound that stabilizes HIFα, thereby decreasing hepcidinexpression in the subject.

In one embodiment, the invention provides a method for treating orpreventing a disorder associated with iron deficiency in a subject, themethod comprising administering to the subject an effective amount of acompound that stabilizes HIFα, thereby treating or preventing thedisorder associated with iron deficiency in the subject. In oneembodiment, the iron deficiency is functional iron deficiency. Invarious embodiments, the disorder is selected from the group consistingof an inflammation, an infection, an immunodeficiency disorder, and aneoplastic disorder; or is selected from the group consisting of anemiaof chronic disease, iron deficiency anemia, and microcytic anemia.

The invention provides a method for enhancing erythropoiesis in asubject having or at risk for having iron deficiency, the methodcomprising administering to the subject an effective amount of acompound that stabilizes HIFα, thereby enhancing erythropoiesis in thesubject. It is contemplated in a certain aspect that the iron deficiencyis functional iron deficiency.

The invention further provides a method for enhancing erythropoiesis ina subject, wherein the subject has or is at risk for having functionaliron deficiency, the method comprising administering to the subject aneffective amount of a compound that stabilizes HIFα, thereby enhancingerythropoiesis in the subject. In various aspects, the chronic diseaseis selected from the group consisting of an inflammation, an infection,an immunodeficiency disorder, and a neoplastic disorder.

A method for enhancing erythropoiesis in a subject, wherein the subjecthas or is at risk for having anemia of chronic disease, is additionallyprovided, the method comprising administering to the subject aneffective amount of a compound that stabilizes HIFα, thereby enhancingerythropoiesis in the subject.

In one embodiment, the invention encompasses a method for enhancingerythropoiesis in a subject wherein the subject is refractory to EPOtherapy, the method comprising administering to the subject an effectiveamount of a compound that stabilizes HIFα, thereby enhancingerythropoiesis in the subject.

A method for treating or preventing anemia of chronic disease in asubject, the method comprising administering to the subject an effectiveamount of a compound that stabilizes HIFα, thereby treating orpreventing anemia of chronic disease in the subject, is also provided.It is contemplated in certain aspects that the anemia of chronic diseaseis associated with a condition selected from the group consisting of aninflammation, an infection, an immunodeficiency disorder, and aneoplastic disorder.

The invention specifically contemplates the following: a method forincreasing reticulocytes in a subject having a chronic disease, themethod comprising administering to the subject an effective amount of acompound that stabilizes HIFα, thereby increasing reticulocytes in thesubject; a method for increasing hematocrit in a subject having achronic disease, the method comprising administering to the subject aneffective amount of a compound that stabilizes HIFα, thereby increasinghematocrit in the subject; a method for increasing hemoglobin in asubject having a chronic disease, the method comprising administering tothe subject an effective amount of a compound that stabilizes HIFα,thereby increasing hemoglobin in the subject; a method for increasingred blood cell count in a subject having a chronic disease, the methodcomprising administering to the subject an effective amount of acompound that stabilizes HIFα, thereby increasing red blood cell countin the subject; a method for increasing mean corpuscular volume in asubject having a chronic disease, the method comprising administering tothe subject an effective amount of a compound that stabilizes HIFα,thereby increasing mean corpuscular volume in the subject; a method forincreasing mean corpuscular hemoglobin in a subject having a chronicdisease, the method comprising administering to the subject an effectiveamount of a compound that stabilizes HIFα thereby increasing meancorpuscular hemoglobin in the subject; a method for increasing serumiron in a subject having a chronic disease, the method comprisingadministering to the subject an effective amount of a compound thatstabilizes HIFα, thereby increasing serum iron in the subject; and amethod or increasing transferrin saturation in a subject having achronic disease, the method comprising administering to the subject aneffective amount of a compound that stabilizes HIFα, thereby increasingtransferrin saturation in the subject. In any one of these methods, thechronic disease is in certain embodiments selected from the groupconsisting of an inflammation, an infection, an immunodeficiencydisorder, and a neoplastic disorder; or is selected from the groupconsisting of anemia of chronic disease, anemia of iron deficiency, irondeficiency, functional iron deficiency, and microcytic anemia.

The following methods are additionally provided: a method for increasingreticulocytes in a subject having iron deficiency, the method comprisingadministering to the subject an effective amount of a compound thatstabilizes HIFα thereby increasing reticulocytes in the subject; amethod for increasing hematocrit in a subject having iron deficiency,the method comprising administering to the subject an effective amountof a compound that stabilizes HIFα, thereby increasing hematocrit in thesubject; a method for increasing hemoglobin in a subject having irondeficiency, the method comprising administering to the subject aneffective amount of a compound that stabilizes HIFα, thereby increasinghemoglobin in the subject; a method for increasing red blood cell countin a subject having iron deficiency, the method comprising administeringto the subject an effective amount of a compound that stabilizes HIFα,thereby increasing red blood cell count in the subject; a method forincreasing mean corpuscular volume in a subject having iron deficiency,the method comprising administering to the subject an effective amountof a compound that stabilizes HIFα, thereby increasing mean corpuscularvolume in the subject; a method for increasing mean corpuscularhemoglobin in a subject having iron deficiency, the method comprisingadministering to the subject an effective amount of a compound thatstabilizes HIFα, thereby increasing mean corpuscular hemoglobin in thesubject; a method for increasing serum iron in a subject having irondeficiency, the method comprising administering to the subject aneffective amount of a compound that stabilizes HIFα, thereby increasingserum iron in the subject; and a method for increasing transferrinsaturation in a subject having iron deficiency, the method comprisingadministering to the subject an effective amount of a compound thatstabilizes HIFα, thereby increasing transferrin saturation in thesubject. In any one of these methods, the iron deficiency in certainembodiments is functional iron deficiency.

The following methods are further contemplated: a method for increasingreticulocytes in a subject having functional iron deficiency, the methodcomprising administering to the subject an effective amount of acompound that stabilizes HIFα, thereby increasing reticulocytes in thesubject; a method for increasing hematocrit in a subject havingfunctional iron deficiency, the method comprising administering to thesubject an effective amount of a compound that stabilizes HIFα, therebyincreasing hematocrit in the subject; a method for increasing hemoglobinin a subject having functional iron deficiency, the method comprisingadministering to the subject an effective amount of a compound thatstabilizes HIFα, thereby increasing hemoglobin in the subject; a methodfor increasing red blood cell count in a subject having functional irondeficiency, the method comprising administering to the subject aneffective amount of a compound that stabilizes HIFα, thereby increasingred blood cell count in the subject; a method for increasing meancorpuscular volume in a subject having functional iron deficiency, themethod comprising administering to the subject an effective amount of acompound that stabilizes HIFα, thereby increasing mean corpuscularvolume in the subject; a method for increasing mean corpuscularhemoglobin in a subject having functional iron deficiency, the methodcomprising administering to the subject an effective amount of acompound that stabilizes HIFα, thereby increasing mean corpuscularhemoglobin in the subject; a method for increasing serum iron in asubject having functional iron deficiency, the method comprisingadministering to the subject an effective amount of a compound thatstabilizes HIFα, thereby increasing serum iron in the subject; and amethod for increasing transferrin saturation in a subject havingfunctional iron deficiency, the method comprising administering to thesubject an effective amount of a compound that stabilizes HIFα, therebyincreasing transferrin saturation in the subject.

In one aspect, the invention includes a method for overcoming orameliorating the consequences of a cytokine-induced impairment oferythropoiesis in a subject, the method comprising administering to thesubject an effective amount of a compound that stabilizes HIFα, therebyovercoming or ameliorating the consequences of the cytokine-inducedimpairment of erythropoiesis in the subject. In various aspects, thecytokine-induced impairment of erythropoiesis is suppression of EPOproduction; or impairment of iron metabolism. In any of theabove-described methods, the cytokine is an inflammatory cytokine. Infurther embodiments, the cytokine is selected from the group consistingof TNF-α, IL-1β, and IFN-γ.

Methods for decreasing cytokine induction of VCAM-1 expression or/andE-selectin expression are also provided, the methods comprisingadministering to a subject in need an effective amount of a compoundthat stabilizes HIFα, thus decreasing cytokine induction of VCAM-1expression or/and E-selectin expression.

In any of the above-described methods, the cytokine is an inflammatorycytokine. In further embodiments, the cytokine is selected from thegroup consisting of TNF-α, IL-1β, and IFN-γ.

Methods for treating or preventing a disorder associated with cytokineactivity in a subject, wherein the disorder is selected from the groupconsisting of iron deficiency, functional iron deficiency, irondeficiency anemia, anemia of chronic disease, and micocytic anemia, areprovided herein, the methods comprising administering to the subject aneffective amount of a compound that stabilizes HIFα, thereby treating orpreventing the disorder associated with cytokine activity. In any of theabove-described methods, the cytokine is an inflammatory cytokine. Infurther embodiments, the cytokine is selected from the group consistingof TNF-α, IL-1β, and IFN-γ.

Methods for treating or preventing a disorder associated with cytokineactivity in a subject, wherein the disorder is associated with acondition selected from the group consisting of an inflammation, aninfection, an immunodeficiency, and a neoplastic disorder, the methodscomprising administering to the subject an effective amount of acompound that stabilizes HIFα, thereby treating or preventing thedisorder associated with cytokine activity, are also provided. In any ofthe above-described methods, the cytokine is an inflammatory cytokine.In further embodiments, the cytokine is selected from the groupconsisting of TNF-α, IL-1β and IFN-γ.

In one aspect, the invention encompasses a method for increasing EPOproduction in the presence of a cytokine in a subject, the methodcomprising administering to the subject an effective amount of acompound that stabilizes HIFα, thereby increasing EPO production in thesubject. A method for treating or preventing microcytosis in a subject,the method comprising administering to the subject an effective amountof a compound that stabilizes HIFα, thereby treating or preventingmicrocytosis in a subject, is also provided herein. In further aspects,the microcytosis is associated with a disorder selected from the groupconsisting of chronic disease, anemia of chronic disease, irondeficiency, functional iron deficiency, and anemia of iron deficiency.In any of the above-described methods, the cytokine is an inflammatorycytokine. In further embodiments, the cytokine is selected from thegroup consisting of TNF-α, IL-1β, and IFN-γ.

In any of the present methods for treating or preventing, it iscontemplated that a compound of the invention can be administered aspart of a combinatorial therapy, additionally comprising administrationof another therapeutic agent, for example, EPO, iron, and vitamins,e.g., B vitamins, etc.

A kit, comprising a compound that stabilizes HIFα and at least one othersupplement is provided herein. In one aspect, the supplement is selectedfrom the group consisting of erythropoietin, iron, and B vitamins, isprovided herein, as is a pharmaceutical composition comprising acompound that stabilizes HIFα and at least one supplement selected fromthe group consisting of erythropoietin, iron, and B vitamins.

The present invention provides compounds and methods for treating orpreventing anemia of chronic disease, wherein the anemia of chronicdisease is associated with increased cytokine levels. In particular, theinvention provides methods and compounds for use in overcoming orameliorating the consequences of cytokine-induced effects in a subjecthaving increased cytokine levels, e.g., cytokine suppression of EPOproduction, cytokine-induced expression of various cell adhesionfactors, etc.

In one embodiment, the invention provides methods and compounds forovercoming cytokine suppression of EPO production. These methods andcompounds are useful in overcoming TNFα and/or IL-1β suppression of EPOproduction, as measured, e.g., by the ability to overcome TNFα and/orIL-1β suppression of EPO production in cultured Hep3B cells.

In one embodiment, the invention provides methods and compounds forreducing cytokine-induced increase in expression of various celladhesion factors. The methods and compounds can be used to overcomeTNFα, IL-1β, and IFN-γ-induced increases in expression of endothelialcell adhesion factors, e.g., VCAM-1 and E-selectin, as measured by,e.g., a decrease in expression level of VCAM-1 or E-selectin inendothelial cells (HUVEC, etc.).

The invention provides methods and compounds for treating or preventingiron deficiency in a subject. In particular, the present methods andcompounds can be used to enhance iron metabolism, or to treat or preventdiseases and disorders associated with impaired iron metabolism, e.g.,impaired iron uptake, storage, processing, transport, mobilization, andutilization, etc.

In one aspect, the methods and compounds modulate expression of factorsinvolved in iron metabolism, e.g., transport, utilization, storage, etc.For example, the methods and compounds increase expression oftransferrin receptor, as measured by, e.g., increased expression oftransferrin receptor in liver cells (e.g., Hep3B, HepG2), kidney cells(e.g., HK-2), or lymphocytes (e.g., THP-1), or by increased solubletransferrin receptor levels in human subjects. The present methods andcompounds increase ceruloplasmin gene expression, as measured, e.g., byincreased gene expression in mouse kidney and in Hep3B cells. In oneaspect, the invention provides methods and compounds that decreasehepcidin gene expression, for example, as measured by reduced geneexpression of hepcidin in mouse liver. In a further aspect, methods andcompounds of the present invention are used to increase expression offactors including NRAMP2, duodenal cytochrome b reductase 1, etc., asmeasured, e.g., by increased gene expression in mouse intestine. Thepresent methods and compounds increase expression of 5-aminolevulinatesynthase, the first enzyme in the heme synthetic pathway andrate-limiting enzyme for heme synthesis, as measured, e.g., by increasedgene expression in mouse intestine.

The present methods and compounds can be used to enhance ironmetabolism. In particular, the present methods and compounds enhanceiron metabolism, as measured by, e.g., increased serum iron levels,increased percent transferrin saturation, and reduced microcytosis in arat model of impaired iron metabolism.

The present invention provides methods and compounds for inducingenhanced erythropoiesis. In particular, the present methods andcompounds enhance erythropoiesis, e.g., as measured by increases inreticulocyte count, hematocrit, and red blood cell count, in a rat modelof impaired erythropoiesis and in human subjects, or as measured by,e.g., increased hemoglobin levels in a rat model of impairederythropoiesis.

The present methods and compounds reduce microcytosis as measured, e.g.,by increased mean corpuscular hemoglobin levels and increased meancorpuscular volume in a rat model of impaired erythropoiesis.

The present methods comprise administering to a subject an effectiveamount of a compound that stabilizes HIFα. Such stabilization can bethrough, e.g., inhibition of HIF hydroxylase activity. A preferredcompound of the invention is a compound that inhibits HIF prolylhydroxylase activity. The inhibition can be direct or indirect, can becompetitive or non-competitive, etc. In various embodiments, a compoundof the invention is selected from the group consisting of 2-oxoglutaratemimetics, iron chelators, and proline analogs. In one aspect, a2-oxoglutarate mimetic is a heterocyclic carbonyl glycine of Formula I,Ia, or Ib. In another aspect, an iron chelator is a hydroxamic acid ofFormula III. In particular embodiments, as exemplified herein, thecompound is Compound D.

Exemplary compounds of the invention include[(1-Chloro-4-hydroxy-isoquinoline-3-carbonyl)-amino]-acetic acid(compound A),[(4-Hydroxy-7-phenoxy-isoquinoline-3-carbonyl)-amino]-acetic acid(compound B),[(4-Hydroxy-7-phenylsulfanyl-isoquinoline-3-carbonyl)-amino]-acetic acid(compound C), and3-{[4-(3,3-Dibenzyl-ureido)-benzenesulfonyl]-[2-(4-methoxy-phenyl)-ethyl]-amino}-N-hydroxy-propionamide(compound D). Additional compounds according to the present inventionand methods for identifying additional compounds of the presentinvention are provided, infra.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B set forth data showing methods and compounds of thepresent invention overcome the suppressive effects of TNF-α on EPOproduction.

FIGS. 2A and 2B set forth data showing methods and compounds of thepresent invention overcome the suppressive effects of TNF-α on EPOproduction in cells pre-treated with TNF-α.

FIGS. 3A and 3B set forth data showing methods and compounds of thepresent invention overcome the suppressive effects of IL-β on EPOproduction.

FIGS. 4A and 4B set forth data showing methods and compounds of thepresent invention overcome the suppressive effects of IL-1β on EPOproduction in cells pre-treated with IL-1β.

FIG. 5 sets forth data showing methods and compounds of the presentinvention reduce VCAM-1 expression associated with TNF-α.

FIGS. 6A, 6B, and 6C set forth data showing increased expression oftransferrin receptor and iron transporter (FIG. 6A), intestinal irontransport protein (FIG. 6B), and 5-aminolevulinate synthase (FIG. 6C)following treatment of mice with compounds of the present invention.

FIG. 7 sets forth data showing methods and compounds of the presentinvention increased reticulocyte counts in an animal model of anemia ofchronic disease.

FIG. 8 sets forth data showing methods and compounds of the presentinvention increased hematocrit in an animal model of anemia of chronicdisease.

FIG. 9 sets forth data showing methods and compounds of the presentinvention increased hemoglobin levels in an animal model of anemia ofchronic disease.

FIG. 10 sets forth data showing methods and compounds of the presentinvention increased red cell count in an animal model of anemia ofchronic disease.

FIG. 11 sets forth data showing methods and compounds of the presentinvention reduced microcytosis in an animal model of anemia of chronicdisease.

FIG. 12 sets forth data showing methods and compounds of the presentinvention increased mean corpuscular hemoglobin and improved hypochromiain an animal model of anemia of chronic disease.

FIG. 13 sets forth data showing methods and compounds of the presentinvention increased hematocrit in normal animals and in an animal modelof anemia of chronic disease.

FIG. 14 sets forth data showing methods and compounds of the presentinvention increased hemoglobin levels in normal animals and in an animalmodel of anemia of chronic disease.

FIG. 15 sets forth data showing methods and compounds of the presentinvention increased red blood cell counts in normal animals and in ananimal model of anemia of chronic disease.

FIG. 16 sets forth data showing methods and compounds of the presentinvention improved mean corpuscular volume in normal animals and in ananimal model of anemia of chronic disease.

FIG. 17 sets forth data showing methods and compounds of the presentinvention improved mean corpuscular hemoglobin levels in normal animalsand in an animal model of anemia of chronic disease.

FIGS. 18A and 18B set forth data showing methods and compounds of thepresent invention increased serum iron levels (FIG. 18A) and transferrinsaturation (FIG. 18B) in normal animals and in an animal model of anemiaof chronic disease.

FIG. 19 sets forth data showing methods and compounds of the presentinvention increased gene expression of NRAMP2 (slc112a) and sproutin(CYBRD1, duodenal cytochrome b reductase 1) in normal animals and in ananimal model of anemia of chronic disease.

FIG. 20 sets forth data showing increased reticulocytes followingadministration of compound of the present invention to healthy humansubjects.

FIG. 21 sets forth data showing increased red blood cell counts inhealthy human subjects administered compound of the present invention.

FIG. 22 sets forth data showing increased soluble transferrin receptorlevels following administration of compound of the present invention tohealthy human subjects.

FIG. 23 sets forth data showing decreased serum ferritin levels inhealthy human subjects administered compound of the present invention.

FIGS. 24A and 24B set forth data showing methods and compounds of thepresent invention reduced VCAM-1 and E-selectin expression associatedwith TNF-α.

FIG. 25 sets forth data showing methods and compounds of the presentinvention reduced VCAM-1 expression associated with TNF-α and IL-1β.

FIG. 26 sets forth data showing methods and compounds of the presentinvention reduced E-selectin expression associated with TNF-α, IL-1β,and IFN-γ.

FIGS. 27A and 27B set forth data showing methods and compounds of thepresent invention and IL-6 synergistically increased EPO levels inhepatocytes.

DESCRIPTION OF THE INVENTION

Before the present compositions and methods are described, it is to beunderstood that the invention is not limited to the particularmethodologies, protocols, cell lines, assays, and reagents described, asthese may vary. It is also to be understood that the terminology usedherein is intended to describe particular embodiments of the presentinvention, and is in no way intended to limit the scope of the presentinvention as set forth in the appended claims.

It must be noted that as used herein and in the appended claims, thesingular forms “a,” “an,” and “the” include plural references unlesscontext clearly dictates otherwise. Thus, for example, a reference to “afragment” includes a plurality of such fragments; a reference to a“compound” is a reference to one of more compounds and to equivalentsthereof as described herein and ask known to those skilled in the art,and so forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of ordinary skillin the art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methods,devices, and materials are now described. All publications cited hereinare incorporated herein by reference in their entirety for the purposeof describing and disclosing the methodologies, reagents, and toolsreported in the publications that might be used in connection with theinvention. Nothing herein is to be construed as an admission that theinvention is not entitled to antedate such disclosure by virtue of priorinvention.

The practice of the present invention will employ, unless otherwiseindicated, conventional methods of chemistry, biochemistry, molecularbiology, cell biology, genetics, immunology and pharmacology, within theskill of the art. Such techniques are explained fully in the literature.See, e.g., Gennaro, A. R., ed. (1990) Remington's PharmaceuticalSciences, 18th ed., Mack Publishing Co.; Hardman, J. G., Limbird, L. E.,and Gilman, A. G., eds. (2001) The Pharmacological Basis ofTherapeutics, 10th ed., McGraw-Hill Co.; Colowick, S. et al., eds.,Methods In Enzymology, Academic Press, Inc.; Weir, D. M., and Blackwell,C. C., eds. (1986) Handbook of Experimental Immunology, Vols. I-IV,Blackwell Scientific Publications; Maniatis, T. et al., eds. (1989)Molecular Cloning: A Laboratory Manual, 2nd edition, Vols. I-III, ColdSpring Harbor Laboratory Press; Ausubel, F. M. et al., eds. (1999) ShortProtocols in Molecular Biology, 4th edition, John Wiley & Sons; Ream etal., eds. (1998) Molecular Biology Techniques: An Intensive LaboratoryCourse, Academic Press; Newton, C. R., and Graham, A., eds. (1997) PCR(Introduction to Biotechniques Series), 2nd ed., Springer Verlag.

DEFINITIONS

The term “anemia of chronic disease” refers to any anemia that developsas a result of, e.g., extended infection, inflammation, neoplasticdisorders, etc. The anemia which develops is often characterized by ashortened red blood cell life span and sequestration of iron inmacrophages, which results in a decrease in the amount of iron availableto make new red blood cells. Conditions associated with anemia ofchronic disease include, but are not limited to, chronic bacterialendocarditis, osteomyelitis, rheumatic fever, ulcerative colitis, andneoplastic disorders. Further conditions include other diseases anddisorders associated with infection, inflammation, and neoplasms,including, e.g., inflammatory infections (e.g., pulmonary abscess,tuberculosis, osteomyelitis, etc.), inflammatory noninfectious disorders(e.g., rheumatoid arthritis, systemic lupus erythrematosus, Crohn'sdisease, hepatitis, inflammatory bowel disease, etc.), and variouscancers, tumors, and malignancies (e.g., carcinoma, sarcoma, lymphoma,etc.).

The terms “disorders” and “diseases” and “conditions” are usedinclusively and refer to any condition deviating from normal.

The term “erythropoietin” refers to any recombinant or naturallyoccurring erythropoietin including, e.g., human erythropoietin (GenBankAccession No. AAA52400; Lin et al. (1985) Proc Natl Acad Sci USA82:7580-7584), EPOETIN human recombinant erythropoietin (Amgen, Inc.,Thousand Oaks Calif.), ARANESP human recombinant erythropoietin (Amgen),PROCRIT human recombinant erythropoietin (Ortho Biotech Products, L. P.,Raritan N.J.), etc.

The term “HIFα” refers to the alpha subunit of hypoxia inducible factorprotein. HIFα may be any human or other mammalian protein, or fragmentthereof, including human HIF-1α (Genbank Accession No. Q16665), HIF-2α(Genbank Accession No. AAB41495), and HIF-3α (Genbank Accession No.AAD22668); murine (Genbank Accession No. Q61221), HIF-2α (GenbankAccession No. BAA20130 and AAB41496), and HIF-3α (Genbank Accession No.AAC72734); rat HIF-1α (Genbank Accession No. CAA70701), HIF-2α (GenbankAccession No. CAB96612), and HIF-3α (Genbank Accession No. CAB96611);and bovine HIF-1α (Genbank Accession No. BAA78675). HIFα may also be anynon-mammalian protein or fragment thereof, including Xenopus laevisHIF-1α (Genbank Accession No. CAB96628), Drosophila melanogaster HIF-1α(Genbank Accession No. JC4851), and chicken HIF-1α (Genbank AccessionNo. BAA34234). HIFα gene sequences may also be obtained by routinecloning techniques, for example by using all or part of a HIFα genesequence described above as a probe to recover and determine thesequence of a HIFα gene in another species.

Fragments of HIFα include the regions defined by human HIF-1α from aminoacid 401 to 603 (Huang et al., supra), amino acid 531 to 575 (Jiang etal. (1997) J Biol Chem 272:19253-19260), amino acid 556 to 575 (Tanimotoet al., supra), amino acid 557 to 571 (Srinivas et al. (1999) BiochemBiophys Res Commun 260:557-561), and amino acid 556 to 575 (Ivan andKaelin (2001) Science 292:464-468). Further, a fragment of HIFα includesany fragment containing at least one occurrence of the motif LXXLAP,e.g., as occurs in the HIFα native sequence at L₃₉₇TLLAP and L₅₅₉EMLAP.Additionally, a fragment of HIFα includes any fragment retaining atleast one functional or structural characteristic of HIFα.

The terms “HIF prolyl hydroxylase” and “HIF PH” refer to any enzymecapable of hydroxylating a proline residue in the HIF protein.Preferably, the proline residue hydroxylated by HIF PH includes theproline found within the motif LXXLAP, e.g., as occurs in the humanHIF-1α native sequence at L₃₉₇TLLAP and L₅₅₉EMLAP. HIF PH includesmembers of the Egl-Nine (EGLN) gene family described by Taylor (2001,Gene 275:125-132), and characterized by Aravind and Koonin (2001, GenomeBiol 2:RESEARCH0007), Epstein et al. (2001, Cell 107:43-54), and Bruickand McKnight (2001, Science 294:1337-1340). Examples of HIF prolylhydroxylase enzymes include human SM-20 (EGLN1) (GenBank Accession No.AAG33965; Dupuy et al. (2000) Genomics 69:348-54), EGLN2 isoform 1(GenBank Accession No. CAC42510; Taylor, supra), EGLN2 isoform 2(GenBank Accession No. NP_(—)060025), and EGLN3 (GenBank Accession No.CAC42511; Taylor, supra); mouse EGLN1 (GenBank Accession No. CAC42515),EGLN2 (GenBank Accession No. CAC42511), and EGLN3 (SM-20) (GenBankAccession No. CAC42517); and rat SM-20 (GenBank Accession No. AAA19321).Additionally, HIF PH may include Caenorhabditis elegans EGL-9 (GenBankAccession No. AAD56365) and Drosophila melanogaster CG1114 gene product(GenBank Accession No. AAF52050). HIF prolyl hydroxylase also includesany fragment of the foregoing full-length proteins that retain at leastone structural or functional characteristic.

The term “prolyl hydroxylase inhibitor” or “PHI,” as used herein, refersto any compound that reduces or otherwise modulates the activity of anenzyme that hydroxylates amino acid residues. Although enzymaticactivity wherein proline residues are hydroxylated is preferred,hydroxylation of other amino acids including, but not limited to,arginine, is also contemplated. Compounds that can be used in themethods of the invention include, for example, iron chelators,2-oxoglutarate mimetics, and modified amino acid, e.g., proline,analogs.

In particular embodiments, the present invention provides for use ofstructural mimetics of 2-oxoglutarate. Such compounds may inhibit thetarget 2-oxoglutarate dioxygenase enzyme family member competitivelywith respect to 2-oxoglutarate and noncompetitively with respect toiron. (Majamaa et al. (1984) Eur J Biochem 138:239-245; and Majamaa etal. (1985) Biochem J 229:127-133.) PHIs specifically contemplated foruse in the present methods are described, e.g., in Majamaa et al.,supra; Kivirikko and Myllyharju (1998) Matrix Biol 16:357-368; Bickel etal. (1998) Hepatology 28:404-411; Friedman et al. (2000) Proc Natl AcadSci USA 97:4736-4741; Franklin (1991) Biochem Soc Trans 19):812 815;Franklin et al. (2001) Biochem J 353:333-338; and InternationalPublication Nos. WO 03/053977 and WO 03/049686, each incorporated byreference herein in its entirety. Exemplary PHIs, including[(1-Chloro-4-hydroxy-isoquinoline-3-carbonyl)-amino]-acetic acid(compound A),[(4-Hydroxy-7-phenoxy-isoquinoline-3-carbonyl)-amino]-acetic acid(compound B),[(4-Hydroxy-7-phenylsulfanyl-isoquinoline-3-carbonyl)-amino]-acetic acid(compound C), and3-{[4-(3,3-Dibenzyl-ureido)-benzenesulfonyl]-[2-(4-methoxy-phenyl)-ethyl]-amino}-N-hydroxy-propionamide(compound D) are used in the present examples to demonstrate the methodsof the invention described herein.

INVENTION

The present invention relates to methods and compounds for inducingenhanced or complete erythropoiesis in a subject. In particular, themethods comprise inducing enhanced or complete erythropoiesis bystabilizing HIFα in a subject. Methods of inducing enhancederythropoiesis by inhibiting HIF prolyl hydroxylase are specificallycontemplated. In specific embodiments, the methods compriseadministering to a subject a compound of the invention. In variousembodiments, the subject can be a cell, tissue, organ, organ system, orwhole organism.

Anemia of chronic disease is the most common form of anemia inhospitalized patients. Anemia of chronic disease occurs in patientshaving inflammatory or malignant disorders, including inflammatoryinfections (e.g., pulmonary abscess, tuberculosis, osteomyelitis, etc.),inflammatory noninfectious disorders (e.g., rheumatoid arthritis,systemic lupus erythrematosus, Crohn's disease, hepatitis, inflammatorybowel disease, etc.), and various cancers, tumors, and malignancies(e.g., carcinoma, sarcoma, lymphoma, etc.), chronic bacterialendocarditis, osteomyelitis, rheumatic fever, ulcerative colitis, andneoplastic disorders.

In one aspect, the invention provides methods for inducing enhanced orcomplete erythropoiesis to treat anemia of chronic disease. Anemia ofchronic disease is associated with numerous chronic disorders,including, for example, rheumatoid arthritis, rheumatic fever,inflammatory bowel disease, ulcerative colitis, systemic lupuserythematosus, vasculitis, neoplastic disorders, etc., as well aschronic infection and chronic inflammation. Reduced or ineffectiveerythropoiesis is a common pathology in patients with anemia of chronicdisease. Reduced or ineffective erythropoiesis can result from variousmetabolic abnormalities in the erythropoietic pathway, including, forexample, suppressed EPO production, decreased EPO responsiveness in thebone marrow, and abnormal iron processing, including, for example,abnormal or ineffective iron uptake, mobilization, storage, andabsorption.

A physiological feature of disorders associated with anemia of chronicdisease is increased production of inflammatory cytokines (Means (1995)Stem Cells 13:32-37 and Means (1999) Int J Hematol 70:7-12), including,for example, tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β),and interferon-γ (IFN-γ), which negatively affect the ability to mediateEPO production, EPO responsiveness, and the coordinate regulation ofiron metabolism. (See, e.g., Roodman et al. (1989) Adv Exp Med Biol271:185-196; Fuchs et al. (1991) Eur J Hematol 46:65-70; Jelkmann et al.(1991) Ann NY Acad Sci 718:300-311; Vannucchi et al. (1994) Br J Hematol87:18-23; and Oldenburg et al. (2001) Aliment Pharmacol Ther15:429-438.) The present invention provides methods for improvingmetabolic and physiologic pathways related to EPO production, EPOsignaling, and iron utilization, resulting in complete or enhancederythropoiesis and reduction or amelioration of anemia of chronicdisease.

The present invention provides advantages over existing therapies foranemia of chronic disease, such as, for example, recombinant EPOadministration. Reduced EPO production is only one aspect of decreasederythropoiesis and it is recognized that administration of recombinantEPO does not address other deficiencies associated with reducederythropoiesis that exist in patients with anemia of chronic disease.(See, e.g., Clibon et al. (1990) Exp Hematol 18:438-441 and Macdougalland Cooper (2002) Neprol Dial Transplant 17(11):39-43.) Thesedeficiencies include, for example, reduced EPO responsiveness of thebone marrow, as well as numerous aspects of iron metabolism thatcontribute to complete or total erythropoiesis, including ironabsorption from the gut, trans-enterocyte transport, oxidation of ironto the ferric state by hephaestin or ceruloplasmin, binding and uptakeof iron by transferrin and transferrin receptor, and iron transport tothe marrow where iron utilization occurs, including heme synthesis. Manypatients are refractory to administration of recombinant EPO for thereasons described above, in which responses to recombinant EPOadministration are reduced or absent, even at high doses of recombinantEPO.

The prevalence of inflammatory cytokines in anemia of chronic diseaseleads to, e.g., decreased serum iron levels and increased iron storage,primarily in macrophages, within a cell compartment not readilyaccessible to emerging erythroid progenitors, which require iron forappropriate heme synthesis. The invention provides methods for enhancingthe metabolic pathways contributing to complete and totalerythropoiesis. In one embodiment, the therapeutic is administered incombination with supplements that further enhance its efficacy, e.g.iron and B vitamins.

Anemia of chronic disease is associated with increased levels offerritin. Despite high levels of ferritin, subjects with anemia ofchronic disease are not able to utilize iron effectively. High levels offerritin are indicative of reduced iron recycling to the marrow andenhanced iron storage, a functional iron deficiency often associatedwith anemia of chronic disease and a pseudo-inflammatory state oftenexisting in uremic chronic kidney disease patients. By decreasingferritin levels, methods and compounds of the present invention decreasestored iron and enhance iron recycling through transferrin andtransferrin receptor. Reduced serum ferritin levels would be indicativeof enhanced iron utilization and enhanced iron recycling to the marrow,thus increasing iron availability for heme production anderythropoiesis.

The genomic response to hypoxia includes changes in gene expression andcell physiology to ameliorate the acute and chronic effects of oxygendeprivation. Hypoxia inducible factor (HIF) is a transcription factorcomposed of an oxygen-regulated alpha subunit (HIFα) and aconstitutively expressed beta subunit (HIFβ). HIFα is destabilized innormoxic environments due to hydroxylation of specific proline residuesby HIF-specific proline hydroxylases (HIF-PHs). However, when oxygenbecomes limiting, e.g., in hypoxic environments, HIF-PH cannothydroxylate HIFα, the subunit is not degraded, and active HIF complexesform, translocate to the nucleus, and activate gene transcription.

In certain aspects, the present invention provides methods treatinganemia of chronic disease by pharmaceutically mimicking hypoxia. Incertain aspects, the methods enhance EPO production in a manner that isresistant to the suppressive effects of inflammatory cytokines. EPOproduction is normally induced by hypoxia or low oxygen but expressionand secretion remain depressed in the presence of inflammatorycytokines, such as TNF-α, IL-1β, and IFN-γ, prevalent in chronic diseasepatients. (See, e.g., Means (1995) Stem Cells 13:32-37; Means (1999) IntJ Hematol 70:7-12; Roodman et al. (1989) Adv Exp Med Biol 271:185-196;Fuchs et al. (1991) Eur J Hematol 46:65-70; Jelkmann et al. (1991) AnnNY Acad Sci 718:300-311; and Vannucchi et al. (1994) Br J Hematol87:18-23.) Prolyl hydroxylase inhibitors overcome the suppressiveeffects of inflammatory cytokines on EPO production, at least in part,as evidenced by the capacity of Hep3B cells to secrete EPO to levelsabove that observed in the presence of inflammatory cytokines. (See,e.g., FIGS. 1A, 1B, 2A, 2B, 3A, 3B, 4A, and 4B.) Agents such as the ironchelator, desferrioxamine, have also shown some efficacy in studies oferythropoietin-resistant anemia, e.g., anemia of chronic disease. (See,e.g., Salvarani et al. (1996) Rheumatol Int 16:45-48 and Goch et al.(1995) Eur J Hematol 55:73-77.)

In other aspects, the present invention provides methods for improvedsignaling by the EPO receptor in the presence of inflammatory cytokines.The prevalence of inflammatory cytokines in chronic disease patientsresults in reduced efficacy of EPO signaling, evidenced by the inabilityof many patients to respond to recombinant EPO with enhancederythropoiesis. This is thought to occur by a decreased sensitivity toEPO bioactivity, as well as defects in bone marrow architecture and/ormicroenvironment. (See, e.g., Clibon et al. (1990) Exp Hematol18:438-441 and Macdougall and Cooper (2002) Neprol Dial Transplant17(11):39-43.) In certain embodiments, the present invention providesmethods for inducing total and complete erythropoiesis by restoring thesensitivity of appropriate cells to signal transduction through the EPOreceptor.

Iron deficiency is one of the most common nutritional deficienciesworldwide and is the leading cause of anemia on a global basis. Ironbalance is fundamentally regulated by the rate of erythropoiesis and thesize of iron stores. Iron deficiency can occur with or without anemia,and has been associated with impaired cognitive development.

Iron deficiency is defined as inadequate iron supply (levels or stores)or as inadequate availability or utilization of iron in the body. Thiscan be due to nutritional deficiencies, e.g., lack of iron in the diet;to iron malabsorption, due, for example, to surgery (post-gastrectomy)or disease (Crohn's disease); or to a depletion in iron supply orincreased iron loss due to chronic or acute blood loss resulting frominjury or trauma, menses, blood donation, phlebotomy (such as due tovarious procedures, surgeries); from increased iron demand, e.g., due torapid growth in infancy or adolescence, pregnancy, erythropoietintherapy, etc.

Iron deficiency can also be functional iron deficiency, e.g., irondeficiency characterized by the subject's impaired ability to access andutilize iron stores. Iron is not available at a rate sufficient to allownormal hemoglobinization of erythrocytes, leading to reducedreticulocyte and erythrocyte cellular hemoglobin content. Functionaliron deficiency is often seen in healthy individuals with apparentlynormal or even increased iron stores but with impaired ironavailability, as measured, e.g., by low levels of percent transferrinsaturation. This type of iron deficiency is frequently associated withacute or with chronic inflammation.

Iron deficiency of any kind can lead to iron-deficient oriron-restricted erythropoiesis, in which red blood cell numbers decreaseand circulating red blood cells are smaller than normal (microcytic) andlack adequate hemoglobin, and as such are pale in color (hypochromic).

Subjects with iron deficiency, including functional iron deficiency, candevelop impaired hemoglobin synthesis, reduced % transferrin saturation,and decreased hemoglobin and hematocrit levels, leading to irondeficiency anemia. Iron deficiency anemia is the most common anemia inthe world. Iron is an essential component of hemoglobin; without iron,the marrow is unable to produce hemoglobin effectively. Iron deficiencyanemia may occur in subjects with depleted or impaired iron supply, ormay occur in subjects having functional iron deficiency, when iron ispresent in storage but is unavailable, e.g., for hemoglobin production.

Iron metabolism encompasses in general the processes by which a cell,tissue, organ, organ system, or whole organism maintains ironhomeostasis by altering, e.g., increasing or decreasing, specificprocesses of iron metabolism. Iron metabolism or iron metabolicprocesses encompass processes involving iron processing, transport,uptake, utilization, storage, mobilization, absorption, etc. Specificaspects of iron metabolism and processing include expression of irontransporters and enzymes which facilitate movement of iron across a cellmembrane and retention or secretion of iron by a cell; alteration inexpression of proteins involved in iron transport in blood; alterationin expression of transferrin and transferrin receptors; alteration inexpression and/or activity of proteins involved in iron absorption;alteration in expression and activity of iron associated transcriptionaland translational regulatory proteins; and alteration of irondistribution within body or culture fluids, including, e.g.,interstitial (i.e. extracellular), intracellular, blood, bone marrow,and the like.

In certain aspects, the present invention provides methods for improvingiron uptake, transport, processing, and utilization. Anemia of chronicdisease is associated with defects in iron utilization that negativelyaffect heme synthesis and hemoglobin formation, resulting in reducederythropoiesis. (See, e.g., Oldenburg et al. (2001) Aliment PharmacolTher 15:429-438.) Decreased serum iron levels, iron mobilization, andany associated increases in iron storage in chronic disease patients,may relate to a microbial defense mechanism of macrophage underconditions of long-lasting inflammation. (See, Fuchs et al. (1991) Eur JHematol 46:65-70.) In some aspects, the present invention providesmethods for increasing effective metabolism of iron by stabilizing HIFα.

Numerous proteins mediate iron metabolism, including proteins such aserythroid 5-aminolevulinate acid synthase (ALAS) (the first andrate-limiting step in heme synthesis) (Bottomley and Muller-Eberhard(1988) Semin Hematol 25:282-302 and Yin et al. (1998) Blood, Cells,Molecules, and Diseases 24(3):41-533), transferrin, transferrinreceptor, iron transporters (involved in iron transport), ceruloplasmin,etc. Increases in transferrin and transferrin receptor expressionstimulate iron uptake by erythroid progenitors and facilitate ironuptake and transport to marrow by macrophage (Goswami et al. (2002)Biochem Cell Biol 80:679-689). Ceruloplasmin increases the oxidation offerrous iron to ferric so that binding to transferrin occurs (Goswami etal. (2002) Biochem Cell Biol 80:679-689). In certain aspects, methods ofthe present invention increase iron metabolism by increasing expressionor activity of proteins involved in iron metabolism, including erythroid5-aminolevulinate synthase, transferrin, transferrin receptor, NRAMP2,sproutin (duodenal cytochrome b reductase 1), and ceruloplasmin. Inother aspects, methods of the present invention increase iron metabolismby decreasing expression or activity hepcidin and by modulatingexpression of ferritin.

In one embodiment, the invention provides methods and compounds forincreasing expression of genes whose products are involved in ironmetabolism and processing, including iron uptake, storage, transport,absorption, etc. Such genes include, but are not limited to, transferrinreceptor, ceruloplasmin, NRAMP2, 5-aminolevulinate synthase, sproutin(CYBRD1), etc. Therapeutic upregulation of genes involved in ironmetabolism and processing will effectively increase iron availabilityand, thereby, produce a beneficial effect in patients with anemia ofchronic disease, anemia of iron deficiency, functional iron deficiency,etc. In another embodiment, the invention provides methods and compoundsfor decreasing expression of hepcidin, a protein associated with ironregulation.

Proper iron metabolism is regulated, in part, by iron response-elementbinding proteins (IRPs), which bind to iron-responsive elements (IREs)found in the 5′- and/or 3′-UTRs of mRNAs encoding, e.g., ferritin (ironstorage), mitochondrial aconitase (energy metabolism),erythroid-aminolevulinate synthase, and transferrin receptor: IRPbinding to a 5′-IRE, as occurs, e.g., in the ferritin transcript,inhibits translation of the mRNA; whereas binding to a 3′-IRE, as occursin, e.g., the transferrin transcript, protects the mRNA fromdegradation. IRP-2 is made constitutively within cells, but is degradedand thus inactivated under iron-replete conditions. IRP-2 is stabilized,however, under iron deplete and/or hypoxic conditions (Hanson et al.(1999) J Biol Chem 274:5047-5052). As IRP-2 decreases expression offerritin, which is responsible for long-term storage of iron, andincreases expression of transferrin and transferrin receptor, IRP-2facilitates iron uptake, transport, and utilization, thus enhancingerythropoiesis (Klausner et al. (1993) Cell 72:19-28). Recently, IREshave been described in other genes that are also necessary forerythropoiesis, including 5-aminolevulinate synthase, the NRAMP2 irontransporter (also known as Slc11a2, DCT1, DMT1, mk (microcytic anemiagene locus in mouse)), and the iron transporter that mediates ironabsorption from dietary sources in the duodenum (Haile (1999) Am J MedSci 318:230-240 and Gunshin et al. (2001) FEBS Lett 509:309-316).

The methods of the present invention, by mimicking conditions ofhypoxia, potentially stabilize IRP-2 in addition to HIFα, thus producinga synergistic effect involving both endogenous EPO production andenhanced iron uptake, transport, and utilization in the production offunctional erythrocytes.

Among adults, iron absorption of dietary iron averages approximately 6%for men and 13% for non-pregnant women. NRAMP2 (also known as DMT1,DCT1, slc11a2) is a ubiquitously expressed divalent metal transporterinvolved in transmembrane transport of non-transferrin bound iron.NRAMP2 is an iron transport protein associated with iron transport fromgastrointestinal lumen into duodenal enterocytes and from erythroblastendosomes to cytoplasm. In animals experiencing dietary iron starvation,NRAMP2 (slc11a2) expression was dramatically increased in the apicalpole of enterocytes in the columnar absorptive epithelium of theproximal duodenum. (See, e.g., Canonne-Hergaux et al. (1999) Blood93:4406-4417.) Genetic rodent models have linked this gene with anemiasassociated with iron deficiency, including hypochromic and microcyticanemic mice (mk mice) having a mutated NRAMP2 gene. MK mice exhibitsevere defects in iron absorption and erythroid iron utilization.

In certain aspects, methods and compounds of the present invention areuseful for increasing iron absorption of dietary iron. The presentinvention provides methods and compounds for increasing expression ofgenes associated with iron transport absorption. In particular,compounds of the present invention were effective at increasingexpression of NRAMP2 in intestine. Increased NRAMP2 (slc11a2) expressionwould be desirable for increasing iron absorption of iron, e.g., dietaryiron, from the gut.

In addition, the present invention provides data showing increasedsproutin gene expression in the intestine of animals treated with acompound of the present invention. Sproutin intestinal iron reductase,also known as Dcytb and Cybrd1 (CYBRD1, duodenal cytochrome b reductase1), is a ferric reductase, and catalyzes the reduction of extracellularferric to ferrous iron associated with iron absorption. Sproutin isco-expressed with NRAMP2 in iron-starved animals in the apical region ofduodenal villi (See, e.g., McKie et al. (2001) Science 291:1755-1759.)

Methods and compounds of the present invention are useful for increasingceruloplasmin gene expression. Ceruloplasmin, also known as aferroxidase-1, converts reduced iron released from storage sites (suchas ferritin) to the oxidized form. Oxidized iron is able to bind to itsplasma transport protein, transferrin. Ceruloplasmin deficiencies areassociated with accumulation of iron in liver and other tissues.Evidence indicates that ceruloplasmin promotes efflux of iron from theliver and promotes influx of iron into iron-deficient cells. (See, e.g.,Tran et al. (2002) J Nutr 132:351-356.)

Compounds of the present invention reduced expression of hepcidin mRNAin mouse liver. Inflammation leads to IL-6 production, which acts onhepatocytes to induce hepcidin production. Hepcidin inhibits macrophageiron release and intestinal iron absorption, reducing iron availabilityand leading to, for example, hypoferremia. Decreased hepcidin expressionis associated with increased iron release from reticuloendothelial cellsand increased intestinal iron absorption. Therefore, methods andcompounds of the present invention are useful for decreasing hepcidinexpression, increasing intestinal iron absorption, and reducinghypoferremia.

Methods for treating anemia associated with hepatitis C virus (HCV)infection are specifically contemplated. Current therapy for HCVinfection include interferon-α and ribaviron in combination. Thiscombination therapy is associated with decreases in hemoglobinconcentrations and anemia. In one aspect, methods and compounds areprovided for treating anemia associated with HCV infection. In anotheraspect, methods and compounds for treating anemia associated withinterferon-α therapy for HCV infection are provided. In another aspect,the present invention provides compounds and methods useful for treatinganemia associated with ribavirin therapy for HCV infection.

Methods for increasing the production of factors required fordifferentiation of erythrocytes from hematopoietic progenitor cellsincluding, e.g., hematopoietic stem cells (HSCs), CFU-GEMM(colony-forming-unit-granulocyte/erythroid/monocyte/megakaryocyte)cells, etc., are also contemplated. Factors that stimulateerythropoiesis include, but are not limited to, erythropoietin. Inanother aspect, the methods increase the production of factors requiredfor iron uptake, transport, and utilization. Such factors include, butare not limited to, erythroid aminolevulinate synthase, transferrin,transferrin receptor, ceruloplasmin, ferritin, etc. In yet anotheraspect, the method increases factors required for differentiation oferythrocytes and additionally factors required for iron uptake,transport, and utilization.

Methods for enhancing responsiveness of hematopoietic precursors toerythropoietin are also contemplated. As described above, suchprecursors include HSCs, CFU-GEMMs, etc. The responsiveness of theprecursor cells can be augmented, e.g., by altering expression oferythropoietin receptors, intracellular factors involved inerythropoietin signaling, and secreted factors that facilitateinteraction of erythropoietin with the receptors. The present inventionprovides methods for enhancing EPO responsiveness of the bone marrow,for example, by increasing EPO receptor expression.

Methods

Various methods are provided herein. In one aspect, the methods compriseadministering to a subject an agent that stabilizes HIFα.

Stabilization of HIFα can be accomplished by any of the methodsavailable to and known by those of skill in the art, and can involve useof any agent that interacts with, binds to, or modifies HIFα or factorsthat interact with HIFα, including, e.g., enzymes for which HIFα is asubstrate. In certain aspects, the present invention contemplatesproviding a constitutively stable HIFα variant, e.g., stable HIFmuteins, etc, or a polynucleotide encoding such a variant. (See, e.g.,U.S. Pat. Nos. 6,562,799 and 6,124,131; and U.S. Pat. No. 6,432,927.) Inother aspects, the present invention contemplates that stabilizing HIFαcomprises administering an agent that stabilizes HIFα. The agent can becomposed of polynucleotides, e.g. antisense sequences (see, e.g.,International Publication No. WO 03/045440); polypeptides; antibodies;other proteins; carbohydrates; fats; lipids; and organic and inorganicsubstances, e.g., small molecules, etc. In a preferred embodiment, thepresent invention contemplates stabilizing HIFα, e.g., in a subject, byadministering to the subject an agent that stabilizes HIFα wherein theagent is a compound, e.g., small molecule compound, etc., thatstabilizes HIFα.

In other embodiments, the methods of the invention comprise stabilizingHIFα by inhibiting the activity of at least one enzyme selected from2-oxoglutarate dioxygenase family. In a preferred embodiment, the enzymeis a HIF hydroxylase enzyme, e.g., EGLN-1, EGLN-2, EGLN-3, etc. (See,e.g., Taylor (2001) Gene 275:125-132; Epstein et al. (2001) Cell107:43-54; and Bruick and McKnight (2001) Science 294:1337-1340.) It isspecifically contemplated, however, that the enzyme be any enzymeselected from the 2-oxoglutarate dioxygenase enzyme family, including,for example, procollagen lysyl hydroxylase, procollagen prolyl3-hydroxylase, procollagen prolyl 4-hydroxylase α(I) and α(II), thymine7-hydroxylase, aspartyl (asparaginyl) β-hydroxylase, ε-N-trimethyllysinehydroxylase, and γ-butyrobetaine hydroxylase, etc. (See, e.g., Majamaaet al. (1985) Biochem J 229:127-133; Myllyharju and Kivirikko (1997)EMBO J 16:1173-1180; Thornburg et al. (1993) 32:14023-14033; and Jia etal. (1994) Proc Natl Acad Sci USA 91:7227-7231.)

In certain embodiments, the methods comprise treating anemia of chronicdisease or regulating iron metabolism by administering to a subject aneffective amount of an agent that stabilizes HIFα. In preferredembodiments, the agent is a compound of the present invention. In oneaspect, the compound stabilizes HIFα by inhibiting the hydroxylation ofcertain residues of HIFα, e.g., proline residues, asparagine residues,etc. In a preferred embodiment, the residues are proline residues. Inspecific embodiments, the residues can be the P₅₆₄ residue in HIF-1α ora homologous proline in another HIFα isoform, or the P₄₀₂ residue inHIF-1α or a homologous proline in another HIFα isoform, etc. In otherembodiments, the present methods may encompass inhibiting hydroxylationof HIFα asparagine residues, e.g., the N₈₀₃ residue of HIF-1α or ahomologous asparagine residue in another HIFα isoform.

Compounds

In preferred methods, the present methods comprise administering to asubject an effective amount of a compound that stabilizes HIFα.Exemplary compounds are disclosed in, e.g., International PublicationNo. WO 03/049686 and International Publication No. WO 03/053997,incorporated herein by reference in their entireties. Specifically,compounds of the invention include the following.

In certain embodiments, a compound of the invention is a compound thatinhibits HIF hydroxylase activity. In various embodiments, the activityis due to a HIF prolyl hydroxylase, such as, for example, EGLN1, EGLN2,or EGLN3, etc. In other embodiments, the activity is due to a HIFasparaginyl hydroxylase, such as, for example, including, but notlimited to, FIH. A preferred compound of the invention is a compoundthat inhibits HIF prolyl hydroxylase activity. The inhibition can bedirect or indirect, can be competitive or non-competitive, etc.

In one aspect, a compound of the invention is any compound that inhibitsor otherwise modulates the activity of a 2-oxoglutarate dioxygenaseenzyme. 2-oxoglutarate dioxygenase enzymes include, but are not limitedto, hydroxylase enzymes. Hydroxylase enzymes hydroxylate targetsubstrate residues and include, for example, prolyl, lysyl, asparaginyl(asparagyl, aspartyl)hydroxylases, etc. Hydroxylases are sometimesdescribed by target substrate, e.g., HIF hydroxylases, procollagenhydroxylases, etc., and/or by targeted residues within the substrate,e.g., prolyl hydroxylases, lysyl hydroxylases, etc., or by both, e.g.,HIF prolyl hydroxylases, procollagen prolyl hydroxylases, etc.Representative 2-oxoglutarate dioxygenase enzymes include, but are notlimited to, HIF hydroxylases, including HIF prolyl hydroxylases, e.g.,EGLN1, EGLN2, and EGLN3, HIF asparaginyl hydroxylases, e.g., factorinhibiting HIF (FIH), etc.; procollagen hydroxylases, e.g., procollagenlysyl hydroxylases, procollagen prolyl hydroxylases, e.g., procollagenprolyl 3-hydroxylase, procollagen prolyl 4-hydroxylase α(I) and α(II),etc.; thymine 7-hydroxylase; aspartyl (asparaginyl) β-hydroxylase;ε-N-trimethyllysine hydroxylase; γ-butyrobetaine hydroxylase, etc.Although enzymatic activity can include any activity associated with any2-oxoglutarate dioxygenase, the hydroxylation of amino acid residueswithin a substrate is specifically contemplated. Although hydroxylationof proline and/or asparagine residues within a substrate is specificallyincluded, hydroxylation of other amino acids is also contemplated.

In one aspect, a compound of the invention that shows inhibitoryactivity toward one or more 2-oxoglutarate dioxygenase enzyme may alsoshow inhibitory activity toward one or more additional 2-oxoglutaratedioxygenase enzymes, e.g., a compound that inhibits the activity of aHIF hydroxylase may additionally inhibit the activity of a collagenprolyl hydroxylase, a compound that inhibits the activity of a HIFprolyl hydroxylase may additionally inhibit the activity of a HIFasparaginyl hydroxylase, etc.

As HIFα is modified by proline hydroxylation, a reaction requiringoxygen and Fe²⁺, the present invention contemplates in one aspect thatthe enzyme responsible for HIFα hydroxylation is a member of the2-oxoglutarate dioxygenase family. Such enzymes include, but are notlimited to, procollagen lysyl hydroxylase, procollagen prolyl3-hydroxylase, procollagen prolyl 4-hydroxylase α(I) and α(II), thymine7-hydroxylase, aspartyl (asparaginyl) β-hydroxylase, ε-N-trimethyllysinehydroxylase, and γ-butyrobetaine hydroxylase, etc. These enzymes requireoxygen, Fe²⁺, 2-oxoglutarate, and ascorbic acid for their hydroxylaseactivity. (See, e.g., Majamaa et al. (1985) Biochem J 229:127-133;Myllyharju and Kivirikko (1997) EMBO J 16:1173-1180; Thornburg et al.(1993) 32:14023-14033; and Jia et al. (1994) Proc Natl Acad Sci USA91:7227-7231.)

In one aspect, a compound of the invention is a compound that stabilizesHIFα. Preferably, the compound stabilizes HIFα through inhibition of HIFhydroxylase activity. It is thus specifically contemplated that acompound of the invention be selected from previously identifiedmodulators of hydroxylase activity. For example, small moleculeinhibitors of prolyl 4-hydroxylase have been identified. (See, e.g.,Majamaa et al. (1984) Eur J Biochem 138:239-245; Majamaa et al. (1985)Biochem J 229:127-133; Kivirikko and Myllyharju (1998) Matrix Biol16:357-368; Bickel et al. (1998) Hepatology 28:404-411; Friedman et al.(2000) Proc Natl Acad Sci USA 97:4736-4741; and Franklin et al. (2001)Biochem J 353:333-338; all incorporated by reference herein in theirentirety.) The present invention contemplates the use of these compoundsin the methods provided herein.

In some aspects, compounds of the present invention include, forexample, structural mimetics of 2-oxoglutarate. Such compounds mayinhibit the target 2-oxoglutarate dioxygenase enzyme family membercompetitively with respect to 2-oxoglutarate and noncompetitively withrespect to iron. (Majamaa et al. (1984) Eur J Biochem 138:239-245; andMajamaa et al. Biochem J 229:127-133.)

In certain embodiments, compounds used in the methods of the inventionare selected from a compound of the formula (I)

whereinA is 1,2-arylidene, 1,3-arylidene, 1,4-arylidene; or (C₁-C₄)-alkylene,optionally substituted by one or two halogen, cyano, nitro,trifluoromethyl, (C₁-C₆)-alkyl, (C₁-C₆)-hydroxyalkyl, (C₁-C₆)-alkoxy,—O—[CH₂]_(x)—C_(f)H_((2f+1−g))Hal_(g), (C₁-C₆)-fluoroalkoxy,(C₁-C₈)-fluoroalkenyloxy, (C₁-C₈)-fluoroalkynyloxy, —OCF₂Cl,—O—CF₂—CHFCl; (C₁-C₆)-alkylmercapto, (C₁-C₆)-alkylsulfinyl,(C₁-C₆)-alkylsulfonyl, (C₁-C₆)-alkylcarbonyl, (C₁-C₆)-alkoxycarbonyl,carbamoyl, N—(C₁-C₄)-alkylcarbamoyl, N,N-di-(C₁-C₄)-alkylcarbamoyl,(C₁-C₆)-alkylcarbonyloxy, (C₃-C₈)-cycloalkyl, phenyl, benzyl, phenoxy,benzyloxy, anilino, N-methylanilino, phenylmercapto, phenylsulfonyl,phenylsulfinyl, sulfamoyl, N—(C₁-C₄)-alkylsulfamoyl,N,N-di-(C₁-C₄)-alkylsulfamoyl; or by a substituted (C₆-C₁₂)-aryloxy,(C₇-C₁₁)-aralkyloxy, (C₆-C₁₂)-aryl, (C₇-C₁₁)-aralkyl radical, whichcarries in the aryl moiety one to five identical or differentsubstituents selected from halogen, cyano, nitro, trifluoromethyl,(C₁-C₆)-alkyl, (C₁-C₆)-alkoxy, —O—[CH₂]_(x)—C_(f)H_((2f+1−g))Hal_(g),—OCF₂Cl, —O—CF₂—CHFCl, (C₁-C₆)-alkylmercapto, (C₁-C₆)-alkylsulfinyl,(C₁-C₆)-alkylsulfonyl, (C₁-C₆)-alkylcarbonyl, (C₁-C₆)-alkoxycarbonyl,carbamoyl, N—(C₁-C₄)-alkylcarbamoyl, N,N-di-(C₁-C₄)-alkylcarbamoyl,(C₁-C₆)-alkylcarbonyloxy, (C₃-C₈)-cycloalkyl, sulfamoyl,N—(C₁-C₄)-alkylsulfamoyl, N,N-di-(C₁-C₄)-alkylsulfamoyl; or wherein A is—CR⁵R⁶ and R⁵ and R⁶ are each independently selected from hydrogen,(C₁-C₆)-alkyl, (C₃-C₇)-cycloalkyl, aryl, or a substituent of theα-carbon atom of an α-amino acid, wherein the amino acid is a naturalL-amino acid or its D-isomer.

B is —CO₂H, —NH₂, —NHSO₂CF₃, tetrazolyl, imidazolyl,3-hydroxyisoxazolyl, —CONHCOR′″, —CONHSOR′″, CONHSO₂R′″, where R′″ isaryl, heteroaryl, (C₃-C₇)-cycloalkyl, or (C₁-C₄)-alkyl, optionallymonosubstituted by (C₆-C₁₂)-aryl, heteroaryl, OH, SH, (C₁-C₄)-alkyl,(C₁-C₄)-alkoxy, (C₁-C₄)-thioalkyl, (C₁-C₄)-sulfinyl, (C₁-C₄)-sulfonyl,CF₃, Cl, Br, F, I, NO2, —COOH, (C₂-C₅)-alkoxycarbonyl, NH₂,mono-(C₁-C₄-alkyl)-amino, di-(C₁-C₄-alkyl)-amino, or(C₁-C₄)-perfluoroalkyl; or wherein B is a CO₂-G carboxyl radical, whereG is a radical of an alcohol G-OH in which G is selected from(C₁-C₂₀)-alkyl radical, (C₃-C₈) cycloalkyl radical, (C₂-C₂₀)-alkenylradical, (C₃-C₈)-cycloalkenyl radical, retinyl radical, (C₂-C₂₀)-alkynylradical, (C₄-C₂₀)-alkenynyl radical, where the alkenyl, cycloalkenyl,alkynyl, and alkenynyl radicals contain one or more multiple bonds;(C₆-C₁₆)-carbocyclic aryl radical, (C₇-C₁₆)-carbocyclic aralkyl radical,heteroaryl radical, or heteroaralkyl radical, wherein a heteroarylradical or heteroaryl moiety of a heteroaralkyl radical contains 5 or 6ring atoms; and wherein radicals defined for G are substituted by one ormore hydroxyl, halogen, cyano, trifluoromethyl, nitro, carboxyl,(C₁-C₁₂)-alkyl, (C₃-C₈)-cycloalkyl, (C₅-C₈)-cycloalkenyl, (C₆-C₁₂)-aryl,(C₇-C₁₆)-aralkyl, (C₂-C₁₂)-alkenyl, (C₂-C₁₂)-alkynyl, (C₁-C₁₂)-alkoxy,(C₁-C₁₂)-alkoxy-(C₁-C₁₂)-alkyl, (C₁-C₁₂)-alkoxy-(C₁-C₁₂)-alkoxy,(C₆-C₁₂)-aryloxy, (C₇-C₁₆)-aralkyloxy, (C₁-C₈)-hydroxyalkyl,—O—[CH₂]_(x)—C_(f)H_((2f+1−g))—F_(g), —OCF₂Cl, —OCF₂—CHFCl,(C₁-C₁₂)-alkylcarbonyl, (C₃-C₈)-cycloalkylcarbonyl,(C₆-C₁₂)-arylcarbonyl, (C₇-C₁₆)-aralkylcarbonyl, cinnamoyl,(C₂-C₁₂)-alkenylcarbonyl, (C₂-C₁₂)-alkynylcarbonyl,(C₁-C₁₂)-alkoxycarbonyl, (C₁-C₁₂)-alkoxy-(C₁-C₁₂)-alkoxycarbonyl,(C₆-C₁₂)-aryloxycarbonyl, (C₇-C₁₆)-aralkoxycarbonyl,(C₃-C₈)-cycloalkoxycarbonyl, (C₂-C₁₂)-alkenyloxycarbonyl,(C₂-C₁₂)-alkynyloxycarbonyl, acyloxy, (C₁-C₁₂)-alkoxycarbonyloxy,(C₁-C₁₂)-alkoxy-(C₁-C₁₂)-alkoxycarbonyloxy, (C₆-C₁₂)-aryloxycarbonyloxy,(C₇-C₁₆) aralkyloxycarbonyloxy, (C₃-C₈)-cycloalkoxycarbonyloxy,(C₂-C₁₂)-alkenyloxycarbonyloxy, (C₂-C₁₂)-alkynyloxycarbonyloxy,carbamoyl, N—(C₁-C₁₂)-alkylcarbamoyl, N,N-di(C₁-C₁₂)-alkylcarbamoyl,N—(C₃-C₈)-cycloalkyl-carbamoyl, N—(C₆-C₁₆)-arylcarbamoyl,N—(C₇-C₁₆)-aralkylcarbamoyl, N—(C₁-C₁₀)-alkyl-N—(C₆-C₁₆)-arylcarbamoyl,N—(C₁-C₁₀)-alkyl-N—(C₇-C₁₆)-aralkylcarbamoyl,N—((C₁-C₁₀)-alkoxy-(C₁-C₁₀)-alkyl)-carbamoyl,N—((C₆-C₁₂)-aryloxy-(C₁-C₁₀)alkyl)-carbamoyl,N—((C₇-C₁₆)-aralkyloxy-(C₁-C₁₀)-alkyl)-carbamoyl,N—(C₁-C₁₀)-alkyl-N—((C₁-C₁₀)-alkoxy-(C₁-C₁₀)-alkyl)-carbamoyl,N—(C₁-C₁₀)-alkyl-N—((C₆-C₁₆)-aryloxy-(C₁-C₁₀)-alkyl)-carbamoyl,N—((C₁-C₁₀)-alkyl-N—((C₇-C₁₆)-aralkyloxy-(C₁-C₁₀)-alkyl)-carbamoyl,carbamoyloxy, N—(C₁-C₁₂)-alkylcarbamoyloxy,N,N-di-(C₁-C₁₂)-alkylcarbamoyloxy, N—(C₃-C₈)-cycloalkylcarbamoyloxy,N—(C₆-C₁₂)-arylcarbamoyloxy, N—(C₇-C₁₆)-aralkylcarbamoyloxy,N—(C₁-C₁₀)-alkyl-N—(C₆-C₁₂)-arylcarbamoyloxy,N(C₁-C₁₀)-alkyl-N—(C₇-C₁₆)-aralkylcarbamoyloxy,N—((C₁-C₁₀)-alkyl)-carbamoyloxy,N—((C₆-C₁₂)-aryloxy-(C₁-C₁₀)-alkyl)-carbamoyloxy,N—((C₇-C₁₆)-aralkyloxy-(C₁-C₁₀)-alkyl)-carbamoyloxy,N—(C₁-C₁₀)-alkyl-N—((C₁-C₁₀)-alkoxy-(C₁-C₁₀)-alkyl)-carbamoyloxy,N—(C₁-C₁₀)-alkyl-N—((C₆-C₁₂)-aryloxy-(C₁-C₁₀)-alkyl)-carbamoyloxy,N—(C₁-C₁₀)-alkyl-N—((C₇-C₁₆)-aralkyloxy-(C₁-C₁₀)-alkyl)-carbamoyloxy,amino, (C₁-C₁₂)-alkylamino, di-(C₁-C₁₂)-alkylamino,(C₃-C₈)-cycloalkylamino, (C₂-C₁₂)-alkenylamino, (C₂-C₁₂)-alkynylamino,N—(C₆-C₁₂)-arylamino, N—(C—C₁₁)-aralkylamino, N-alkyl-aralkylamino,N-alkyl-arylamino, (C₁-C₁₂)-alkoxyamino,(C₁-C₁₂)-alkoxy-N—(C₁-C₁₀)-alkylamino, (C₁-C₁₂)-alkylcarbonylamino,(C₃-C₈)-cycloalkylcarbonylamino, (C₆-C₁₂) arylcarbonylamino,(C₇-C₁₆)-aralkylcarbonylamino,(C₁-C₁₂)-alkylcarbonyl-N—(C₁-C₁₀)-alkylamino,(C₃-C₈)-cycloalkylcarbonyl-N—(C₁-C₁₀)-alkylamino,(C₆-C₁₂)-arylcarbonyl-N—(C₁-C₁₀)alkylamino,(C₇-C₁₁)-aralkylcarbonyl-N—(C₁-C₁₀)-alkylamino,(C₁-C₁₂)-alkylcarbonylamino-(C₁-C₈)-alkyl,(C₃-C₈)-cycloalkylcarbonylamino-(C₁-C₈)alkyl,(C₆-C₁₂)-arylcarbonylamino-(C₁-C₈)-alkyl,(C₇-C₁₂)-aralkylcarbonylamino(C₁-C₈)-alkyl, amino-(C₁-C₁₀)-alkyl,N—(C₁-C₁₀) alkylamino-(C₁-C₁₀)-alkyl,N,N-di-(C₁-C₁₀)-alkylamino-(C₁-C₁₀)-alkyl,(C₃-C₈)cycloalkylamino-(C₁-C₁₀)-alkyl, (C₁-C₁₂)-alkylmercapto,(C₁-C₁₂)-alkylsulfinyl, (C₁-C₁₂)-alkylsulfonyl, (C₆-C₁₆)-arylmercapto,(C₆-C₁₆)-arylsulfinyl, (C₆-C₁₂)-arylsulfonyl, (C₇-C₁₆)-aralkylmercapto,(C₇-C₁₆)-aralkylsulfinyl, (C₇-C₁₆)-aralkylsulfonyl, sulfamoyl,N—(C₁-C₁₀)-alkylsulfamoyl, N,N-di(C₁-C₁₀)-alkylsulfamoyl,(C₃-C₈)-cycloalkylsulfamoyl, N—(C₆-C₁₂)-alkylsulfamoyl,N—(C₇-C₁₆)-aralkylsulfamoyl, N—(C₁-C₁₀)-alkyl-N—(C₆-C₁₂)-arylsulfamoyl,N—(C₁-C₁₀)-alkyl-N—(C₇-C₁₆)-aralkylsulfamoyl, (C₁-C₁₀)-alkylsulfonamido,N—(C₁-C₁₀)-alkyl)-(C₁-C₁₀)-alkylsulfonamido,(C₇-C₁₆)-aralkylsulfonamido, orN—((C₁-C₁₀)-alkyl-(C₇-C₁₆)-aralkylsulfonamido; wherein radicals whichare aryl or contain an aryl moiety, may be substituted on the aryl byone to five identical or different hydroxyl, halogen, cyano,trifluoromethyl, nitro, carboxyl, (C₁-C₁₂)-alkyl, (C₃-C₈)-cycloalkyl,(C₆-C₁₂)-aryl, (C₇-C₁₆)-aralkyl, (C₁-C₁₂)-alkoxy,(C₁-C₁₂)-alkoxy-(C₁-C₁₂)alkyl, (C₁-C₁₂)-alkoxy-(C₁ C₁₂)alkoxy,(C₆-C₁₂)-aryloxy, (C₇-C₁₆)-aralkyloxy, (C₁-C₈)-hydroxyalkyl,(C₁-C₁₂)-alkylcarbonyl, (C₃-C₈)-cycloalkyl-carbonyl,(C₆-C₁₂)-arylcarbonyl, (C₇-C₁₆)aralkylcarbonyl, (C₁-C₁₂)-alkoxycarbonyl,(C₁-C₁₂)-alkoxy-(C₁-C₁₂)-alkoxycarbonyl, (C₆-C₁₂)-aryloxycarbonyl,(C₇-C₁₆)-aralkoxycarbonyl, (C₃-C₈)-cycloalkoxycarbonyl,(C₂-C₁₂)-alkenyloxycarbonyl, (C₂-C₁₂)-alkynyloxycarbonyl,(C₁-C₁₂)-alkylcarbonyloxy, (C₃-C₈)-cycloalkylcarbonyloxy,(C₆-C₁₂)-arylcarbonyloxy, (C₇-C₁₆)-aralkylcarbonyloxy, cinnamoyloxy,(C₂-C₁₂)-alkenylcarbonyloxy, (C₂-C₁₂)-alkynylcarbonyloxy,(C₁-C₁₂)-alkoxycarbonyloxy, (C₁-C₁₂)-alkoxy-(C₁-C₁₂)-alkoxycarbonyloxy,(C₆-C₁₂)-aryloxycarbonyloxy, (C₇-C₁₆)-aralkyloxycarbonyloxy,(C₃-C₈)-cycloalkoxycarbonyloxy, (C₂-C₁₂)-alkenyloxycarbonyloxy,(C₂-C₁₂)-alkynyloxycarbonyloxy, carbamoyl, N—(C₁-C₁₂)-alkylcarbamoyl,N,N-di-(C₁-C₁₂)-alkylcarbamoyl, N—(C₃-C₈)-cycloalkylcarbamoyl,N—(C₆-C₁₂)-arylcarbamoyl, N—(C₇-C₁₆)-aralkylcarbamoyl,N—(C₁-C₁₀)-alkyl-N—(C₆-C₁₂)-arylcarbamoyl,N—(C₁-C₁₀)-alkyl-N—(C₇-C₁₆)-aralkylcarbamoyl,N—((C₁-C₁₀)-alkoxy-(C₁-C₁₀)-alkyl)-carbamoyl,N—((C₆-C₁₂)-aryloxy-(C₁-C₁₀)-alkyl)-carbamoyl,N—((C₇-C₁₆)-aralkyloxy-(C₁-C₁₀)-alkyl)-carbamoyl,N—(C₁-C₁₀)-alkyl-N—((C₁-C₁₀)-alkoxy-(C₁-C₁₀)-alkyl)-carbamoyl,N—(C₁-C₁₀)-alkyl-N—((C₆-C₁₂)-aryloxy-(C₁-C₁₀)-alkyl)-carbamoyl,N—(C₁-C₁₀-alkyl-N—((C₇-C₁₆)-aralkyloxy-(C₁-C₁₀)-alkyl)-carbamoyl,carbamoyloxy, N—(C₁-C₁₂)-alkylcarbamoyloxy,N,N-di-(C₁-C₁₂)-alkylcarbamoyloxy, N—(C₃-C₈)-cycloalkylcarbamoyloxy,N—(C₆-C₁₂)-arylcarbamoyloxy, N—(C₇-C₁₆)-aralkylcarbamoyloxy,N—(C₁-C₁₀)-alkyl-N—(C₆-C₁₂)-arylcarbamoyloxy,N(C₁-C₁₀)-alkyl-N—(C₇-C₁₆)-aralkylcarbamoyloxy,N—((C₁-C₁₀)-alkyl)-carbamoyloxy,N—((C₆-C₁₂)-aryloxy-(C₁-C₁₀)-alkyl)-carbamoyloxy,N—((C₇-C₁₆)-aralkyloxy-(C₁-C₁₀)-alkyl)-carbamoyloxy,N—(C₁-C₁₀)-alkyl-N—((C₁-C₁₀)-alkoxy-(C₁-C₁₀)-alkyl)-carbamoyloxy,N—(C₁-C₁₀)-alkyl-N—((C₆-C₁₂)-aryloxy-(C₁-C₁₀)-alkyl)-carbamoyloxy,N—(C₁-C₁₀)-alkyl-N—((C₇-C₁₆)-aralkyloxy-(C₁-C₁₀)-alkyl)-carbamoyloxy,amino, (C₁-C₁₂)-alkylamino, di-(C₁-C₁₂)-alkylamino,(C₃-C₈)-cycloalkylamino, (C₃-C₁₂)-alkenylamino, (C₃-C₁₂)-alkynylamino,N—(C₆-C₁₂)-arylamino, N—(C₇-C₁₁)-aralkylamino, N-alkylaralkylamino,N-alkyl-arylamino, (C₁-C₁₂)-alkoxyamino,(C₁-C₁₂)-alkoxy-N—(C₁-C₁₀)-alkylamino, (C₁-C₁₂)-alkylcarbonylamino,(C₃-C₈)-cycloalkylcarbonylamino, (C₆-C₁₂)-arylcarbonylamino,(C₇-C₁₆)-alkylcarbonylamino,(C₁-C₁₂)-alkylcarbonyl-N—(C₁-C₁₀)-alkylamino,(C₃-C₈)-cycloalkylcarbonyl-N—(C₁-C₁₀)-alkylamino,(C₆-C₁₂)-arylcarbonyl-N—(C₁-C₁₀)-alkylamino,(C₇-C₁₁)-aralkylcarbonyl-N—(C₁-C₁₀)-alkylamino,(C₁-C₁₂)-alkylcarbonylamino-(C₁-C₈)-alkyl,(C₃-C₈)-cycloalkylcarbonylamino-(C₁-C₈)-alkyl,(C₆-C₁₂)-arylcarbonylamino-(C₁-C₈)-alkyl,(C₇-C₁₆)-aralkylcarbonylamino-(C₁-C₈)-alkyl, amino-(C₁-C₁₀)-alkyl,N—(C₁-C₁₀)-alkylamino-(C₁-C₁₀)alkyl,N,N-di-(C₁-C₁₀)-alkylamino-(C₁-C₁₀)-alkyl,(C₃-C₈)-cycloalkylamino-(C₁-C₁₀)-alkyl, (C₁-C₁₂)-alkylmercapto,(C₁-C₁₂)-alkylsulfinyl, (C₁-C₁₂)-alkylsulfonyl, (C₆-C₁₂)-arylmercapto,(C₆-C₁₂)-arylsulfinyl, (C₆-C₁₂)-arylsulfonyl, (C₇-C₁₆)-aralkylmercapto,(C₇-C₁₆)-aralkylsulfinyl, or (C₇-C₁₆)-aralkylsulfonyl;

X is O or S;

Q is O, S, NR′, or a bond;

where, if Q is a bond, R⁴ is halogen, nitrile, or trifluoromethyl;

or where, if Q is O, S, or NR′, R⁴ is hydrogen, (C₁-C₁₀)-alkyl radical,(C₂-C₁₀)-alkenyl radical, (C₂-C₁₀)-alkynyl radical, wherein alkenyl oralkynyl radical contains one or two C—C multiple bonds; unsubstitutedfluoroalkyl radical of the formula —[CH₂]_(x)—C_(f)H_((2f+1−g))—F_(g),(C₁-C₈)-alkoxy-(C₁-C₆)-alkyl radical,(C₁-C₆)-alkoxy-(C₁-C₄)-alkoxy-(C₁-C₄)-alkyl radical, aryl radical,heteroaryl radical, (C₇-C₁₁)-aralkyl radical, or a radical of theformula Z—[CH₂]_(v)—[O]_(w)—[CH₂]_(t)-E  (Z)whereE is a heteroaryl radical, a (C₃-C₈)-cycloalkyl radical, or a phenylradical of the formula F

v is 0-6,w is 0 or 1,t is 0-3, andR⁷, R⁸, R⁹, R¹⁰, and R¹¹ are identical or different and are hydrogen,halogen, cyano, nitro, trifluoromethyl, (C₁-C₆)-alkyl,(C₃-C₈)-cycloalkyl, (C₁-C₆)-alkoxy,—O—[CH₂]_(x)—C_(f)H_((2f+1−g))—F_(g), —O—CF₂—Cl, —O—CF₂—CHFCl,(C₁-C₆)-alkylmercapto, (C₁-C₆)-hydroxyalkyl,(C₁-C₆)-alkoxy-(C₁-C₆)-alkoxy, (C₁-C₆)-alkoxy-(C₁-C₆)-alkyl,(C₁-C₆)-alkylsulfinyl, (C₁-C₆)-alkylsulfonyl, (C₁-C₆)-alkylcarbonyl,(C₁-C₈)-alkoxycarbonyl, carbamoyl, N—(C₁-C₈)-alkylcarbamoyl,N,N-di-(C₁-C₈)-alkylcarbamoyl, or (C₇-C₁₁)-aralkylcarbamoyl, optionallysubstituted by fluorine, chlorine, bromine, trifluoromethyl,(C₁-C₆)-alkoxy, N—(C₃-C₈)-cycloalkylcarbamoyl,N—(C₃-C₈)-cycloalkyl-(C₁-C₄)-alkylcarbamoyl, (C₁-C₆)-alkylcarbonyloxy,phenyl, benzyl, phenoxy, benzyloxy, NR^(Y)R^(Z) wherein R^(y) and R^(z)are independently selected from hydrogen, (C₁-C₁₂)-alkyl,(C₁-C₈)-alkoxy-(C₁-C₈)-alkyl, (C₇-C₁₂)-aralkoxy-(C₁-C₈)-alkyl,(C₆-C₁₂)-aryloxy-(C₁-C₈)-alkyl, (C₃-C₁₀)-cycloalkyl, (C₃-C₁₂)-alkenyl,(C₃-C₁₂)-alkynyl, (C₆-C₁₂)-aryl, (C₇-C₁₁)-aralkyl, (C₁-C₁₂)-alkoxy,(C₇-C₁₂)aralkoxy, (C₁-C₁₂)-alkylcarbonyl, (C₃-C₈)-cycloalkylcarbonyl,(C₆-C₁₂) arylcarbonyl, (C₇-C₁₆)-aralkylcarbonyl; or further whereinR^(y) and R^(z) together are —[CH2]_(h), in which a CH₂ group can bereplaced by O, S, N—(C₁-C₄)-alkylcarbonylimino, orN—(C₁-C₄)-alkoxycarbonylimino; phenylmercapto, phenylsulfonyl,phenylsulfinyl, sulfamoyl, N—(C₁-C₈)-alkylsulfamoyl, orN,N-di-(C₁-C₈)-alkylsulfamoyl; or alternatively R⁷ and R⁸, R⁸ and R⁹, R⁹and R¹⁰, or R¹⁰ and R¹¹, together are a chain selected from —[CH₂]_(n)—or —CH═CH—CH═CH—, where a CH₂ group of the chain is optionally replacedby O, S, SO, SO₂, or NR^(Y); and n is 3, 4, or 5; and if E is aheteroaryl radical, said radical can carry 1-3 substituents selectedfrom those defined for R⁷-R¹¹, or if E is a cycloalkyl radical, theradical can carry one substituent selected from those defined forR⁷-R¹¹;or where, if Q is NR′, R⁴ is alternatively R″, where R′ and R″ areidentical or different and are hydrogen, (C₆-C₁₂)-aryl,(C₇-C₁₁)-aralkyl, (C₁-C₈)-alkyl, (C₁-C₈)-alkoxy-(C₁-C₈)-alkyl,(C₇-C₁₂)-aralkoxy-(C₁-C₈)-alkyl, (C₆-C₁₂)-aryloxy-(C₁-C₈)-alkyl,(C₁-C₁₀)-alkylcarbonyl, optionally substituted (C₇-C₁₆)-aralkylcarbonyl,or optionally substituted C₆-C₁₂)-arylcarbonyl; or R′ and R″ togetherare —[CH₂]_(h), in which a CH₂ group can be replaced by O, S,N-acylimino, or N—(C₁-C₁₀)-alkoxycarbonylimino, and h is 3 to 7.Y is N or CR³;R¹, R² and R³ are identical or different and are hydrogen, hydroxyl,halogen, cyano, trifluoromethyl, nitro, carboxyl, (C₁-C₂₀)-alkyl,(C₃-C₈)-cycloalkyl, (C₃-C₈)cycloalkyl-(C₁-C₁₂)-alkyl,(C₃-C₈)-cycloalkoxy, (C₃-C₈)-cycloalkyl-(C₁-C₁₂)-alkoxy,(C₃-C₈)-cycloalkyloxy-(C₁-C₁₂)-alkyl,(C₃-C₈)-cycloalkyloxy-(C₁-C₁₂)-alkoxy,(C₃-C₈)-cycloalkyl-(C₁-C₈)-alkyl-(C₁-C₆)-alkoxy,(C₃-C₈)-cycloalkyl-(C₁-C₈)-alkoxy-(C₁-C₆)-alkyl,(C₃-C₈)-cycloalkyloxy-(C₁-C₈)-alkoxy-(C₁-C₆)-alkyl,(C₃-C₈)-cycloalkoxy-(C₁-C₈)-alkoxy-(C₁-C₈)-alkoxy, (C₆-C₁₂)-aryl,(C₇-C₁₆)-aralkyl, (C₇-C₁₆)-aralkenyl, (C₇-C₁₆)-aralkynyl,(C₂-C₂₀)-alkenyl, (C₂-C₂₀)-alkynyl, (C₁-C₂₀)-alkoxy,(C₂-C₂₀)-alkenyloxy, (C₂-C₂₀)-alkynyloxy, retinyloxy,(C₁-C₂₀)-alkoxy-(C₁-C₁₂)-alkyl, (C₁-C₁₂)-alkoxy-(C₁-C₁₂)-alkoxy,(C₁-C₁₂)-alkoxy-(C₁-C₈)-alkoxy-(C₁-C₈)-alkyl, (C₆-C₁₂)-aryloxy,(C₇-C₁₆)-aralkyloxy, (C₆-C₁₂)-aryloxy-(C₁-C₆)-alkoxy,(C₇-C₁₆)-aralkoxy-(C₁-C₆)-alkoxy, (C₁-C₁₆)-hydroxyalkyl,(C₆-C₁₆)-aryloxy-(C₁-C₈)-alkyl, (C₇-C₁₆)-aralkoxy-(C₁-C₈)-alkyl,(C₆-C₁₂)-aryloxy-(C₁-C₈)-alkoxy-(C₁-C₆)-alkyl,(C₇-C₁₂)-aralkyloxy-(C₁-C₈)-alkoxy-(C₁-C₆)-alkyl,(C₂-C₂₀)-alkenyloxy-(C₁-C₆)-alkyl, (C₂-C₂₀)-alkynyloxy-(C₁-C₆)-alkyl,retinyloxy-(C₁-C₆)-alkyl, —O—[CH₂]_(x)CfH_((2f+1−g))—F_(g), —OCF₂Cl,—OCF₂—CHFCl, (C₁-C₂₀)-alkylcarbonyl, (C₃-C₈)-cycloalkylcarbonyl,(C₆-C₁₂)-arylcarbonyl, (C₇-C₁₆)-aralkylcarbonyl, cinnamoyl,(C₂-C₂₀)-alkenylcarbonyl, (C₂-C₂₀)-alkynylcarbonyl,(C₁-C₂₀)-alkoxycarbonyl, (C₁-C₁₂)-alkoxy-(C₁-C₁₂)-alkoxycarbonyl,(C₆-C₁₂)-aryloxycarbonyl, (C₇-C₁₆)-aralkoxycarbonyl,(C₃-C₈)-cycloalkoxycarbonyl, (C₂-C₂₀)-alkenyloxycarbonyl,retinyloxycarbonyl, (C₂-C₂₀)-alkynyloxycarbonyl,(C₆-C₁₂)-aryloxy-(C₁-C₆)-alkoxycarbonyl,(C₇-C₁₆)-aralkoxy-(C₁-C₆)-alkoxycarbonyl,(C₃-C₈)-cycloalkyl-(C₁-C₆)-alkoxycarbonyl,(C₃-C₈)-cycloalkoxy-(C₁-C₆)-alkoxycarbonyl, (C₁-C₁₂)-alkylcarbonyloxy,(C₃-C₈)-cycloalkylcarbonyloxy, (C₆-C₁₂)-arylcarbonyloxy,(C₇-C₁₆)-aralkylcarbonyloxy, cinnamoyloxy, (C₂-C₁₂)-alkenylcarbonyloxy,(C₂-C₁₂)-alkynylcarbonyloxy, (C₁-C₁₂)-alkoxycarbonyloxy,(C₁-C₁₂)-alkoxy-(C₁-C₁₂)-alkoxycarbonyloxy, (C₆-C₁₂)-aryloxycarbonyloxy,(C₇-C₁₆)-aralkyloxycarbonyloxy, (C₃-C₈)-cycloalkoxycarbonyloxy,(C₂-C₁₂)-alkenyloxycarbonyloxy, (C₂-C₁₂)-alkynyloxycarbonyloxy,carbamoyl, N—(C₁-C₁₂)-alkylcarbamoyl, N,N-di-(C₁-C₁₂)-alkylcarbamoyl,N—(C₃-C₈)-cycloalkylcarbamoyl, N,N-dicyclo-(C₃-C₈)-alkylcarbamoyl,N—(C₁-C₁₀)-alkyl-N—(C₃-C₈)-cycloalkylcarbamoyl,N—((C₃-C₈)-cycloalkyl-(C₁-C₆)-alkyl)-carbamoyl,N—(C₁-C₆)-alkyl-N—((C₃-C₈)-cycloalkyl-(C₁-C₆)-alkyl)-carbamoyl,N-(+)-dehydroabietylcarbamoyl,N—(C₁-C₆)-alkyl-N-(+)-dehydroabietylcarbamoyl, N—(C₆-C₁₂)-arylcarbamoyl,N—(C₇-C₁₆)-aralkylcarbamoyl, N—(C₁-C₁₀)-alkyl-N—(C₆-C₁₆)-arylcarbamoyl,N—(C₁-C₁₀)-alkyl-N—((C₇-C₁₆)-aralkylcarbamoyl,N—((C₁-C₁₈)-alkoxy-(C₁-C₁₀)-alkyl)-carbamoyl,N—((C₆-C₁₆)-aryloxy-(C₁-C₁₀)-alkyl)-carbamoyl,N—((C₇-C₁₆)-aralkyloxy-(C₁-C₁₀)-alkyl)-carbamoyl,N—(C₁-C₁₀)-alkyl-N—((C₁-C₁₀)-alkoxy-(C₁-C₁₀)-alkyl)-carbamoyl,N—(C₁-C₁₀)-alkyl-N—((C₆-C₁₂)-aryloxy-(C₁-C₁₀)-alkyl)-carbamoyl,N—(C₁-C₁₀)-alkyl-N—((C₇-C₁₆)-aralkyloxy-(C₁-C₁₀)-alkyl)-carbamoyl;CON(CH₂)_(h), in which a CH₂ group can be replaced by O, S,N—(C₁-C₈)-alkylimino, N—(C₃-C₈)-cycloalkylimino,N—(C₃-C₈)-cycloalkyl-(C₁-C₄)-alkylimino, N—(C₆-C₁₂)-arylimino,N—(C₇-C₁₆)-aralkylimino, N—(C₁-C₄)-alkoxy-(C₁-C₆)-alkylimino, and h isfrom 3 to 7; a carbamoyl radical of the formula R

in whichR^(x) and R^(v) are each independently selected from hydrogen,(C₁-C₆)-alkyl, (C₃-C₇)-cycloalkyl, aryl, or the substituent of anα-carbon of an α-amino acid, to which the L- and D-amino acids belong,s is 1-5,T is OH, or NR*R**, and R*, R** and R*** are identical or different andare selected from hydrogen, (C₆-C₁₂)-aryl, (C₇-C₁₁)-aralkyl,(C₁-C₈)-alkyl, (C₃-C₈)-cycloalkyl, (+)-dehydroabietyl,(C₁-C₈)-alkoxy-(C₁-C₈)-alkyl, (C₇-C₁₂)-aralkoxy-(C₁-C₈)-alkyl,(C₆-C₁₂)-aryloxy-(C₁-C₈)-alkyl, (C₁-C₁₀)-alkanoyl, optionallysubstituted (C₇-C₁₆)-aralkanoyl, optionally substituted (C₆-C₁₂)-aroyl;or R* and R** together are —[CH₂]_(h), in which a CH₂ group can bereplaced by O, S, SO, SO₂, N-acylamino, N—(C₁-C₁₀)-alkoxycarbonylimino,N—(C₁-C₈)-alkylimino, N—(C₃-C₈)-cycloalkylimino,N—(C₃-C₈)-cycloalkyl-(C₁-C₄)-alkylimino, N—(C₆-C₁₂)-arylimino,N—(C₇-C₁₆)-aralkylimino, N—(C₁-C₄)-alkoxy-(C₁-C₆)-alkylimino, and h isfrom 3 to 7; carbamoyloxy, N—(C₁-C₁₂)-alkylcarbamoyloxy,N,N-di-(C₁-C₁₂)-alkylcarbamoyloxy, N—(C₃-C₈)-cycloalkylcarbamoyloxy,N—(C₆-C₁₂)-arylcarbamoyloxy, N—(C₇-C₁₆)-aralkylcarbamoyloxy,N—(C₁-C₁₀)-alkyl-N—(C₆-C₁₂)-arylcarbamoyloxy,N—(C₁-C₁₀)-alkyl-N—(C₇-C₁₆)-aralkylcarbamoyloxy,N—((C₁-C₁₀)-alkyl)-carbamoyloxy,N—((C₆-C₁₂)-aryloxy-(C₁-C₁₀)-alkyl)-carbamoyloxy,N—((C₇-C₁₆)-aralkyloxy-(C₁-C₁₀)-alkyl)-carbamoyloxy,N—(C₁-C₁₀)-alkyl-N—((C₁-C₁₀)-alkoxy-(C₁-C₁₀)-alkyl)-carbamoyloxy,N—(C₁-C₁₀)-alkyl-N—((C₆-C₁₂)-aryloxy-(C₁-C₁₀)-alkyl)-carbamoyloxy,N—(C₁-C₁₀)-alkyl-N—((C₇-C₁₆)-aralkyloxy-(C₁-C₁₀)-alkyl)-carbamoyloxyamino,(C₁-C₁₂)-alkylamino, di-(C₁-C₁₂)-alkylamino, (C₃-C₈)-cycloalkylamino,(C₃-C₁₂)-alkenylamino, (C₃-C₁₂)-alkynylamino, N—(C₆-C₁₂)-arylamino,N—(C₇-C₁₁)-aralkylamino, N-alkyl-aralkylamino, N-alkyl-arylamino,(C₁-C₁₂)-alkoxyamino, (C₁-C₁₂)-alkoxy-N—(C₁-C₁₀)-alkylamino,(C₁-C₁₂)-alkanoylamino, (C₃-C₈)-cycloalkanoylamino, (C₆-C₁₂)-aroylamino,(C₇-C₁₆)-aralkanoylamino, (C₁-C₁₂)-alkanoyl-N—(C₁-C₁₀)-alkylamino,(C₃-C₈)-cycloalkanoyl-N—(C₁-C₁₀)-alkylamino,(C₆-C₁₂)-aroyl-N—(C₁-C₁₀)-alkylamino,(C₇-C₁₁)-aralkanoyl-N—(C₁-C₁₀)-alkylamino,(C₁-C₁₂)-alkanoylamino-(C₁-C₈)-alkyl,(C₃-C₈)-cycloalkanoylamino-(C₁-C₈)-alkyl,(C₆-C₁₂)-aroylamino-(C₁-C₈)-alkyl,(C₇-C₁₆)-aralkanoylamino-(C₁-C₈)-alkyl, amino-(C₁-C₁₀)-alkyl,N—(C₁-C₁₀)-alkylamino-(C₁-C₁₀)-alkyl,N,N-di(C₁-C₁₀)-alkylamino-(C₁-C₁₀)-alkyl,(C₃-C₈)-cycloalkylamino(C₁-C₁₀)-alkyl, (C₁-C₂₀)-alkylmercapto,(C₁-C₂₀)-alkylsulfinyl, (C₁-C₂₀)-alkylsulfonyl, (C₆-C₁₂)-arylmercapto,(C₆-C₁₂)-arylsulfinyl, (C₆-C₁₂)-arylsulfonyl, (C₇-C₁₆)-aralkylmercapto,(C₇-C₁₆)-aralkylsulfinyl, (C₇-C₁₆)-aralkylsulfonyl,(C₁-C₁₂)-alkylmercapto-(C₁-C₆)-alkyl,(C₁-C₁₂)-alkylsulfinyl-(C₁-C₆)-alkyl,(C₁-C₁₂)-alkylsulfonyl-(C₁-C₆)-alkyl,(C₆-C₁₂)-arylmercapto-(C₁-C₆)-alkyl,(C₆-C₁₂)-arylsulfinyl-(C₁-C₆)-alkyl,(C₆-C₁₂)-arylsulfonyl-(C₁-C₆)-alkyl,(C₇-C₁₆)-aralkylmercapto-(C₁-C₆)-alkyl,(C₇-C₁₆)-aralkylsulfinyl-(C₁-C₆)-alkyl,(C₇-C₁₆)-aralkylsulfonyl-(C₁-C₆)-alkyl, sulfamoyl,N—(C₁-C₁₀)-alkylsulfamoyl, N,N-di-(C₁-C₁₀)-alkylsulfamoyl,(C₃-C₈)-cycloalkylsulfamoyl, N—(C₆-C₁₂)-arylsulfamoyl,N—(C₇-C₁₆)-aralkylsulfamoyl, N—(C₁-C₁₀)-alkyl-N—(C₆-C₁₂)-arylsulfamoyl,N—(C₁-C₁₀)-alkyl-N—(C₇-C₁₆)-aralkylsulfamoyl, alkylsulfonamido,N—((C₁-C₁₀)-alkyl)-(C₁-C₁₀)-alkylsulfonamido,(C₇-C₁₆)-aralkylsulfonamido, andN—((C₁-C₁₀)-alkyl-(C₇-C₁₆)-aralkylsulfonamido; where an aryl radical maybe substituted by 1 to 5 substituents selected from hydroxyl, halogen,cyano, trifluoromethyl, nitro, carboxyl, (C₂-C₁₆)-alkyl,(C₃-C₈)-cycloalkyl, (C₃-C₈)-cycloalkyl-(C₁-C₁₂)-alkyl,(C₃-C₈)-cycloalkoxy, (C₃-C₈)-cycloalkyl-(C₁-C₁₂)-alkoxy,(C₃-C₈)-cycloalkyloxy-(C₁-C₁₂)-alkyl,(C₃-C₈)-cycloalkyloxy-(C₁-C₁₂)-alkoxy,(C₃-C₈)-cycloalkyl-(C₁-C₈)-alkyl-(C₁-C₆)-alkoxy,(C₃-C₈)-cycloalkyl(C₁-C₈)-alkoxy-(C₁-C₆)-alkyl,(C₃-C₈)-cycloalkyloxy-(C₁-C₈)-alkoxy-(C₁-C₆)-alkyl,(C₃-C₈)-cycloalkoxy-(C₁-C₈)-alkoxy-(C₁-C₈)-alkoxy, (C₆-C₁₂)-aryl,(C₇-C₁₆)-aralkyl, (C₂-C₁₆)-alkenyl, (C₂-C₁₂)-alkynyl, (C₁-C₁₆)-alkoxy,(C₁-C₁₆)-alkenyloxy, (C₁-C₁₂)-alkoxy-(C₁-C₁₂)-alkyl,(C₁-C₁₂)-alkoxy-(C₁-C₁₂)-alkoxy,(C₁-C₁₂)-alkoxy(C₁-C₈)-alkoxy-(C₁-C₈)-alkyl, (C₆-C₁₂)-aryloxy,(C₇-C₁₆)-aralkyloxy, (C₆-C₁₂)-aryloxy-(C₁-C₆)-alkoxy,(C₇-C₁₆)-aralkoxy-(C₁-C₆)-alkoxy, (C₁-C₈)-hydroxyalkyl,(C₆-C₁₆)-aryloxy-(C₁-C₈)-alkyl, (C₇-C₁₆)-aralkoxy-(C₁-C₈)-alkyl,(C₆-C₁₂)-aryloxy-(C₁-C₈)-alkoxy-(C₁-C₆)-alkyl,(C₇-C₁₂)-aralkyloxy-(C₁-C₈)-alkoxy-(C₁-C₆)-alkyl,—O—[CH₂]_(x)C_(f)H_((2f+−g))F_(g), —OCF₂Cl, —OCF₂—CHFCl,(C₁-C₁₂)-alkylcarbonyl, (C₃-C₈)-cycloalkylcarbonyl,(C₆-C₁₂)-arylcarbonyl, (C₇-C₁₆)-aralkylcarbonyl,(C₁-C₁₂)-alkoxycarbonyl, (C₁-C₁₂)-alkoxy-(C₁-C₁₂)-alkoxycarbonyl,(C₆-C₁₂)-aryloxycarbonyl, (C₇-C₁₆)-aralkoxycarbonyl,(C₃-C₈)-cycloalkoxycarbonyl, (C₂-C₁₂)-alkenyloxycarbonyl,(C₂-C₁₂)-alkynyloxycarbonyl, (C₆-C₁₂)-aryloxy-(C₁-C₆)-alkoxycarbonyl,(C₇-C₁₆)-aralkoxy-(C₁-C₆)-alkoxycarbonyl,(C₃-C₈)-cycloalkyl-(C₁-C₆)-alkoxycarbonyl,(C₃-C₈)-cycloalkoxy-(C₁-C₆)-alkoxycarbonyl, (C₁-C₁₂)-alkylcarbonyloxy,(C₃-C₈)-cycloalkylcarbonyloxy, (C₆-C₁₂)-arylcarbonyloxy,(C₇-C₁₆)-aralkylcarbonyloxy, cinnamoyloxy, (C₂-C₁₂)-alkenylcarbonyloxy,(C₂-C₁₂)-alkynylcarbonyloxy, (C₁-C₁₂)-alkoxycarbonyloxy,(C₁-C₁₂)-alkoxy-(C₁-C₁₂)-alkoxycarbonyloxy, (C₆-C₁₂)-aryloxycarbonyloxy,(C₇-C₁₆)-aralkyloxycarbonyloxy, (C₃-C₈)-cycloalkoxycarbonyloxy,(C₂-C₁₂)-alkenyloxycarbonyloxy, (C₂-C₁₂)-alkynyloxycarbonyloxy,carbamoyl, N—(C₁-C₁₂)-alkylcarbamoyl, N,N-di(C₁-C₁₂)-alkylcarbamoyl,N—(C₃-C₈)-cycloalkylcarbamoyl, N,N-dicyclo-(C₃-C₈)-alkylcarbamoyl,N—(C₁-C₁₀)-alkyl-N—(C₃-C₈)-cycloalkylcarbamoyl,N—((C₃-C₈)-cycloalkyl-(C₁-C₆)-alkyl)carbamoyl,N—(C₁-C₆)-alkyl-N—((C₃-C₈)-cycloalkyl-(C₁-C₆)-alkyl)carbamoyl,N-(+)-dehydroabietylcarbamoyl,N—(C₁-C₆)-alkyl-N-(+)-dehydroabietylcarbamoyl, N—(C₆-C₁₂)-arylcarbamoyl,N—(C₇-C₁₆)-aralkylcarbamoyl, N—(C₁-C₁₀-alkyl-N—(C₆-C₁₆)-arylcarbamoyl,N—(C₁-C₁₀)-alkyl-N—(C₇-C₁₆)-aralkylcarbamoyl,N—((C₁-C₁₆)-alkoxy-(C₁-C₁₀)-alkyl)carbamoyl,N—((C₆-C₁₆)-aryloxy-(C₁-C₁₀)-alkyl)carbamoyl,N—((C₇-C₁₆)-aralkyloxy-(C₁-C₁₀-alkyl)carbamoyl,N—(C₁-C₁₀)-alkyl-N—((C₁-C₁₀)-alkoxy-(C₁-C₁₀)-alkyl)carbamoyl,N—(C₁-C₁₀)-alkyl-N—((C₆-C₁₂)-aryloxy-(C₁-C₁₀)-alkyl)carbamoyl,N—(C₁-C₁₀)-alkyl-N—((C₇-C₁₆)-aralkyloxy-(C₁-C₁₀)-alkyl)-carbamoyl,CON(CH₂)_(h), in which a CH₂ group can be replaced by, O, S,N—(C₁-C₈)-alkylimino, N—(C₃-C₈)-cycloalkylimino,N—(C₃-C₈)-cycloalkyl-(C₁-C₄)-alkylimino, N—(C₆-C₁₂)-arylimino,N—(C₇-C₁₆)-aralkylimino, N—(C₁-C₄)-alkoxy-(C₁-C₆)-alkylimino, and h isfrom 3 to 7; carbamoyloxy, N—(C₁-C₁₂)-alkylcarbamoyloxy,N,N-di-(C₁-C₁₂)-alkylcarbamoyloxy, N—(C₃-C₈)-cycloalkylcarbamoyloxy,N—(C₆-C₁₆)-arylcarbamoyloxy, N—(C₇-C₁₆)-aralkylcarbamoyloxy,N—(C₁-C₁₀)-alkyl-N—(C₆-C₁₂)-arylcarbamoyloxy,N—(C₁-C₁₀)-alkyl-N—(C₇-C₁₆)-aralkylcarbamoyloxy,N—((C₁-C₁₀)-alkyl)carbamoyloxy,N—((C₆-C₁₂)-aryloxy-(C₁-C₁₀)-alkyl)carbamoyloxy,N—((C₇-C₁₆)-aralkyloxy-(C₁-C₁₀)-alkyl)carbamoyloxy,N—(C₁-C₁₀)-alkyl-N—((C₁-C₁₀)-alkoxy-(C₁-C₁₀)-alkyl)carbamoyloxy,N—(C₁-C₁₀)-alkyl-N—((C₆-C₁₂)-aryloxy-(C₁-C₁₀)-alkyl)carbamoyloxy,N—(C₁-C₁₀)-alkyl-N—((C₇-C₁₆)-aralkyloxy-(C₁-C₁₀-alkyl)carbamoyloxy,amino, (C₁-C₁₂)-alkylamino, di-(C₁-C₁₂)-alkylamino,(C₃-C₈)-cycloalkylamino, (C₃-C₁₂)-alkenylamino, (C₃-C₁₂)-alkynylamino,N—(C₆-C₁₂)-arylamino, N—(C₇-C₁₁)-aralkylamino, N-alkyl-aralkylamino,N-alkyl-arylamino, (C₁-C₁₂)-alkoxyamino,(C₁-C₁₂)-alkoxy-N—(C₁-C₁₀)-alkylamino, (C₁-C₁₂)-alkanoylamino,(C₃-C₈)-cycloalkanoylamino, (C₆-C₁₂)-aroylamino,(C₇-C₁₆)-aralkanoylamino, (C₁-C₁₂)-alkanoyl-N—(C₁-C₁₀)-alkylamino,(C₃-C₈)-cycloalkanoyl-N—(C₁-C₁₀)-alkylamino,(C₆-C₁₂)-aroyl-N—(C₁-C₁₀)-alkylamino,(C₇-C₁₁)-aralkanoyl-N—(C₁-C₁₀)-alkylamino,(C₁-C₁₂)-alkanoylamino-(C₁-C₈)-alkyl,(C₃-C₈)-cycloalkanoylamino-(C₁-C₈)-alkyl,(C₆-C₁₂)-aroylamino-(C₁-C₈)-alkyl,(C₇-C₁₆)-aralkanoylamino-(C₁-C₈)-alkyl, amino-(C₁-C₁₀)-alkyl,N—(C₁-C₁₀)-alkylamino-(C₁-C₁₀)-alkyl,N,N-di-(C₁-C₁₀)-alkylamino-(C₁-C₁₀)-alkyl,(C₃-C₈)-cycloalkylamino-(C₁-C₁₀)-alkyl, (C₁-C₁₂)-alkylmercapto,(C₁-C₁₂)-alkylsulfinyl, (C₁-C₁₂)-alkylsulfonyl, (C₆-C₁₆)-arylmercapto,(C₆-C₁₆)-arylsulfinyl, (C₆-C₁₆)-arylsulfonyl, (C₇-C₁₆)-aralkylmercapto,(C₇-C₁₆)-aralkylsulfinyl, or (C₇-C₁₆)-aralkylsulfonyl;or wherein R¹ and R², or R² and R³ form a chain [CH₂]_(o), which issaturated or unsaturated by a C═C double bond, in which 1 or 2 CH₂groups are optionally replaced by O, S, SO, SO₂, or NR′, and R′ ishydrogen, (C₆-C₁₂)-aryl, (C₁-C₈)-alkyl, (C₁-C₈)-alkoxy-(C₁-C₈)-alkyl,(C₇-C₁₂)-aralkoxy-(C₁-C₈)-alkyl, (C₆-C₁₂)-aryloxy-(C₁-C₈)-alkyl,(C₁-C₁₀)-alkanoyl, optionally substituted (C₇-C₁₆)-aralkanoyl, oroptionally substituted (C6-C12)-aroyl; and o is 3, 4 or 5;or wherein the radicals R¹ and R², or R² and R³, together with thepyridine or pyridazine carrying them, form a5,6,7,8-tetrahydroisoquinoline ring, a 5,6,7,8-tetrahydroquinoline ring,or a 5,6,7,8-tetrahydrocinnoline ring;or wherein R¹ and R², or R² and R³ form a carbocyclic or heterocyclic 5-or 6-membered aromatic ring;or where R¹ and R², or R² and R³, together with the pyridine orpyridazine carrying them, form an optionally substituted heterocyclicring systems selected from thienopyridines, furanopyridines,pyridopyridines, pyrimidinopyridines, imidazopyridines,thiazolopyridines, oxazolopyridines, quinoline, isoquinoline, andcinnoline; where quinoline, isoquinoline or cinnoline preferably satisfythe formulae Ia, Ib and Ic:

and the substituents R¹² to R²³ in each case independently of each otherhave the meaning of R¹, R² and R³;or wherein the radicals R¹ and R², together with the pyridine carryingthem, form a compound of Formula Id:

where V is S, O, or NR^(k), and R^(k) is selected from hydrogen,(C₁-C₆)-alkyl, aryl, or benzyl; where an aryl radical may be optionallysubstituted by 1 to 5 substituents as defined above; andR²⁴, R²⁵, R²⁶, and R²⁷ in each case independently of each other have themeaning of R¹, R² and R³;f is 1 to 8;g is 0 or 1 to (2f+1);x is 0 to 3; andh is 3 to 7;including the physiologically active salts and prodrugs derivedtherefrom.

Exemplary compounds according to Formula (I) are described in EuropeanPatent Nos. EP0650960 and EP0650961. All compounds listed in EP0650960and EP0650961, in particular, those listed in the compound claims andthe final products of the working examples, are hereby incorporated intothe present application by reference herein. Exemplary compounds ofFormula (I) include, but are not limited to,[(3-Hydroxy-pyridine-2-carbonyl)-amino]-acetic acid and[(3-methoxy-pyridine-2-carbonyl)-amino]-acetic acid.

Additionally, exemplary compounds according to Formula (I) are describedin U.S. Pat. No. 5,658,933. All compounds listed in U.S. Pat. No.5,658,933, in particular, those listed in the compound claims and thefinal products of the working examples, are hereby incorporated into thepresent application by reference herein. Exemplary compounds of Formula(I) include, but are not limited to, 3-methoxypyridine-2-carboxylic acidN-(((hexadecyloxy)-carbonyl)-methyl)-amide hydrochloride,3-methoxypyridine-2-carboxylic acidN-(((1-octyloxy)-carbonyl)-methyl)-amide, 3-methoxypyridine-2-carboxylicacid N-(((hexyloxy)-carbonyl)-methyl)-amide,3-methoxypyridine-2-carboxylic acidN-(((butyloxy)-carbonyl)-methyl)-amide, 3-methoxypyridine-2-carboxylicacid N-(((2-nonyloxy)-carbonyl)-methyl)-amide racemate,3-methoxypyridine-2-carboxylic acidN-(((heptyloxy)-carbonyl)-methyl)-amide,3-benzyloxypyridine-2-carboxylic acidN-(((octyloxy)-carbonyl)-methyl)-amide, 3-benzyloxypyridine-2-carboxylicacid N-(((butyloxy)-carbonyl)-methyl)-amide,5-(((3-(1-butyloxy)-propyl)-amino)-carbonyl)-3-methoxypyridine-2-carboxylicacid N-((benzyloxycarbonyl)-methyl)-amide,5-(((3-(1-butyloxy)-propyl)-amino)-carbonyl)-3-methoxypyridine-2-carboxylicacid N-(((1-butyloxy)-carbonyl)-methyl)-amide, and5-(((3-lauryloxy)-propyl)amino)-carbonyl)-3-methoxypyridine-2-carboxylicacid N-(((benzyloxy)-carbonyl)-methyl)-amide.

Additional compounds according to Formula (I) are substitutedheterocyclic carboxyamides described in U.S. Pat. No. 5,620,995;3-hydroxypyridine-2-carboxamidoesters described in U.S. Pat. No.6,020,350; sulfonamidocarbonylpyridine-2-carboxamides described in U.S.Pat. No. 5,607,954; and sulfonamidocarbonyl-pyridine-2-carboxamides andsulfonamidocarbonyl-pyridine-2-carboxamide esters described in U.S. Pat.Nos. 5,610,172 and 5,620,996. All compounds listed in these patents, inparticular, those compounds listed in the compound claims and the finalproducts of the working examples, are hereby incorporated into thepresent application by reference herein.

Exemplary compounds according to Formula (Ia) are described in U.S. Pat.Nos. 5,719,164 and 5,726,305. All compounds listed in the foregoingpatents, in particular, those listed in the compound claims and thefinal products of the working examples, are hereby incorporated into thepresent application by reference herein. Exemplary compounds of Formula(Ia) include, but are not limited to,N-((3-hydroxy-6-isopropoxy-quinoline-2-carbonyl)-amino)-acetic acid,N-((6-(1-butyloxy)-3-hydroxyquinolin-2-yl)-carbonyl)-glycine,[(3-hydroxy-6-trifluoromethoxy-quinoline-2-carbonyl)-amino]-acetic acid,N-((6-chloro-3-hydroxyquinolin-2-yl)-carbonyl)-glycine,N-((7-chloro-3-hydroxyquinolin-2-yl)-carbonyl)-glycine, and[(6-chloro-3-hydroxy-quinoline-2-carbonyl)-amino]-acetic acid.

Exemplary compounds according to Formula (Ib) are described in U.S. Pat.No. 6,093,730. All compounds listed in U.S. Pat. No. 6,093,730, inparticular, those listed in the compound claims and the final productsof the working examples, are hereby incorporated into the presentapplication by reference herein. Exemplary compounds of Formula (Ib)include, but are not limited to, N-((1-chloro-4-hydroxy-7-(2-propyloxy)isoquinolin-3-yl)-carbonyl)-glycine,N-((1-chloro-4-hydroxy-6-(2-propyloxy)isoquinolin-3-yl)-carbonyl)-glycine,N-((1-chloro-4-hydroxy-isoquinoline-3-carbonyl)-amino)-acetic acid(compound A),N-((1-chloro-4-hydroxy-7-methoxyisoquinolin-3-yl)-carbonyl)-glycine,N-((1-chloro-4-hydroxy-6-methoxyisoquinolin-3-yl)-carbonyl)-glycine,N-((7-butyloxy)-1-chloro-4-hydroxyisoquinolin-3-yl)-carbonyl)-glycine,N-((6-benzyloxy-1-chloro-4-hydroxy-isoquinoline-3-carbonyl)-amino)-aceticacid,((7-benzyloxy-1-chloro-4-hydroxy-isoquinoline-3-carbonyl)-amino)-aceticacid methyl ester,N-((7-benzyloxy-1-chloro-4-hydroxy-isoquinoline-3-carbonyl)-amino)-aceticacid, N-((8-chloro-4-hydroxyisoquinolin-3-yl)-carbonyl)-glycine,N-((7-butoxy-4-hydroxy-isoquinoline-3-carbonyl)-amino)-acetic acid.

Additionally, compounds related to Formula (I) that can also be used inthe methods of the invention include, but are not limited to,6-cyclohexyl-1-hydroxy-4-methyl-1H-pyridin-2-one,7-(4-methyl-piperazin-1-ylmethyl)-5-phenylsulfanylmethyl-quinolin-8-ol,4-nitro-quinolin-8-ol, 5-butoxymethyl-quinolin-8-ol,[(4-Hydroxy-7-phenoxy-isoquinoline-3-carbonyl)-amino]-acetic acid(compound B), and[(4-Hydroxy-7-phenylsulfanyl-isoquinoline-3-carbonyl)-amino]-acetic acid(compound C). Further, the invention provides additional exemplarycompounds wherein, e.g., position A and B together may be, e.g.,hexanoic acid, cyanomethyl, 2-aminoethyl, benzoic acid,1H-benzoimidazol-2-ylmethyl, etc.

In other embodiments, compounds used in the methods of the invention areselected from a compound of the formula (III)

or pharmaceutically acceptable salts thereof, wherein:a is an integer from 1 to 4;b is an integer from 0 to 4;c is an integer from 0 to 4;Z is selected from the group consisting of (C₃-C₁₀) cycloalkyl, (C₃-C₁₀)cycloalkyl independently substituted with one or more Y¹, 3-10 memberedheterocycloalkyl and 3-10 membered heterocycloalkyl independentlysubstituted with one or more Y¹; (C₅-C₂₀) aryl, (C₅-C₂₀) arylindependently substituted with one or more Y¹, 5-20 membered heteroaryland 5-20 membered heteroaryl independently substituted with one or moreY¹;Ar¹ is selected from the group consisting of (C₅-C₂₀) aryl, (C₅-C₂₀)aryl independently substituted with one or more Y², 5-20 memberedheteroaryl and 5-20 membered heteroaryl independently substituted withone or more Y²;each Y¹ is independently selected from the group consisting of alipophilic functional group, (C₅-C₂₀) aryl, (C₆-C₂₆) alkaryl, 5-20membered heteroaryl and 6-26 membered alk-heteroaryl;each Y² is independently selected from the group consisting of —R′,—OR′, —OR″, —SR′, —SR″, —NR′R′, —NO₂, —CN, -halogen, -trihalomethyl,trihalomethoxy, —C(O)R′, —C(O)OR′, —C(O)NR′R′, —C(O)NR′OR′,—C(NR′R′)═NOR′, —NR′—C(O)R′, —SO₂R′, —SO₂R″, —NR′—SO₂—R′,—NR′—C(O)—NR′R′, tetrazol-5-yl, —NR′—C(O)—OR′, —C(NR′R′)═NR′, —S(O)—R′,—S(O)—R″, and —NR′—C(S)—NR′R′; andeach R′ is independently selected from the group consisting of —H,(C₁-C₈) alkyl, (C₂-C₈) alkenyl, and (C₂-C₈) alkynyl; andeach R″ is independently selected from the group consisting of (C₅-C₂₀)aryl and (C₅-C₂₀) aryl independently substituted with one or more —OR′,—SR′, —NR′R′, —NO₂, —CN, halogen or trihalomethyl groups,or wherein c is 0 and Ar¹ is an N′ substituted urea-aryl, the compoundhas the structural formula (IIIa):

or pharmaceutically acceptable salts thereof, wherein:a, b, and Z are as defined above; andR³⁵ and R³⁶ are each independently selected from the group consisting ofhydrogen, (C₁-C₈) alkyl, (C₂-C₈) alkenyl, (C₂-C₈) alkynyl, (C₃-C₁₀)cycloalkyl, (C₅-C₂₀) aryl, (C₅-C₂₀) substituted aryl, (C₆-C₂₆) alkaryl,(C₆-C₂₆) substituted alkaryl, 5-20 membered heteroaryl, 5-20 memberedsubstituted heteroaryl, 6-26 membered alk-heteroaryl, and 6-26 memberedsubstituted alk-heteroaryl; andR³⁷ is independently selected from the group consisting of hydrogen,(C₁-C₈) alkyl, (C₂-C₈) alkenyl, and (C₂-C₈) alkynyl.

Exemplary compounds of Formula (III) are described in InternationalPublication No. WO 00/50390. All compounds listed in WO 00/50390, inparticular, those listed in the compound claims and the final productsof the working examples, are hereby incorporated into the presentapplication by reference herein. Exemplary compounds of Formula (III)include3-{[4-(3,3-dibenzyl-ureido)-benzenesulfonyl]-[2-(4-methoxy-phenyl)-ethyl]-amino}-N-hydroxy-propionamide(compound D),3-{{4-[3-(4-chloro-phenyl)-ureido]-benzenesulfonyl}-[2-(4-methoxy-phenyl)-ethyl]-amino}-N-hydroxy-propionamide,and3-{{4-[3-(1,2-diphenyl-ethyl)-ureido]-benzenesulfonyl}-[2-(4-methoxy-phenyl)-ethyl]-amino}-N-hydroxy-propionamide.

Methods for identifying compounds of the invention are also provided. Incertain aspects, a compound of the invention is one that stabilizesHIFα. The ability of a compound to stabilize or activate HIFα can bemeasured, for example, by direct measurement of HIFα in a sample,indirect measurement of HIFα, e.g., by measuring a decrease in HIFαassociated with the von Hippel Lindau protein (see, e.g., InternationalPublication No. WO 00/69908), or activation of HIF responsive targetgenes or reporter constructs (see, e.g., U.S. Pat. No. 5,942,434).Measuring and comparing levels of HIF and/or HIF-responsive targetproteins in the absence and presence of the compound will identifycompounds that stabilize HIFα and/or activate HIF.

In other aspects, a compound of the invention is one that inhibits HIFhydroxylase activity. Assays for hydroxylase activity are standard inthe art. Such assays can directly or indirectly measure hydroxylaseactivity. For example, an assay can measure hydroxylated residues, e.g.,proline, asparagine, etc., present in the enzyme substrate, e.g., atarget protein, a synthetic peptide mimetic, or a fragment thereof.(See, e.g., Palmerini et al. (1985) J Chromatogr 339:285-292.) Areduction in hydroxylated residue, e.g., proline or asparagine, in thepresence of a compound is indicative of a compound that inhibitshydroxylase activity. Alternatively, assays can measure other productsof the hydroxylation reaction, e.g., formation of succinate from2-oxoglutarate. (See, e.g., Cunliffe et al. (1986) Biochem J240:617-619.) Kaule and Gunzler (1990; Anal Biochem 184:291-297)describe an exemplary procedure that measures production of succinatefrom 2-oxoglutarate.

Procedures such as those described above can be used to identifycompounds that modulate HIF hydroxylase activity. Target protein mayinclude HIFα or a fragment thereof, e.g., HIF(556-575). Enzyme mayinclude, e.g., HIF prolyl hydroxylase (see, e.g., GenBank Accession No.AAG33965, etc.) or HIF asparaginyl hydroxylase (see, e.g., GenBankAccession No. AAL27308, etc.), obtained from any source. Enzyme may alsobe present in a crude cell lysate or in a partially purified form. Forexample, procedures that measure HIF hydroxylase activity are describedin Ivan et al. (2001, Science 292:464-468; and 2002, Proc Natl Acad SciUSA 99:13459-13464) and Hirsila et al. (2003, J Biol Chem278:30772-30780); additional methods are described in InternationalPublication No. WO 03/049686. Measuring and comparing enzyme activity inthe absence and presence of the compound will identify compounds thatinhibit hydroxylation of HIFα.

A compound of the invention is one that further produces a measurableeffect, as measured in vitro or in vivo, as demonstrated by enhancederythropoiesis, enhanced iron metabolism, or therapeutic improvement ofconditions including, e.g., iron deficiency, including functional irondeficiency; anemia of chronic disease, iron deficiency, and microcytosisor microcytic anemia; or a condition associated with inflammation,infection, immunodeficiency, or neoplastic disorder.

The measurable effect can be any one of the following parameters:increased hemoglobin, hematocrit, reticulocyte, red blood cell count,plasma EPO, etc.; improved iron metabolism, as measured by lessening ofobserved symptoms, including, e.g., mitigation of chronic fatigue,pallor, dizziness, etc., or by increased serum iron levels, alteredserum ferritin levels, % transferrin saturation, total iron bindingcapacity, improved reticulocyte counts, hemoglobin, hematocrit, e.g.,all as measured by standard blood count analysis.

Pharmaceutical Formulations and Routes of Administration

The compositions of the present invention can be delivered directly orin pharmaceutical compositions containing excipients, as is well knownin the art. Present methods of treatment can comprise administration ofan effective amount of a compound of the present invention to a subjecthaving or at risk for a metabolic disorder; particularly a disorderassociated with glucose regulation, e.g., diabetes, hyperglycemia, etc.In a preferred embodiment, the subject is a mammalian subject, and in amost preferred embodiment, the subject is a human subject.

An effective amount, e.g., dose, of compound or drug can readily bedetermined by routine experimentation, as can an effective andconvenient route of administration and an appropriate formulation.Various formulations and drug delivery systems are available in the art.(See, e.g., Gennaro, ed. (2000) Remington's Pharmaceutical Sciences,supra; and Hardman, Limbird, and Gilman, eds. (2001) The PharmacologicalBasis of Therapeutics, supra.)

Suitable routes of administration may, for example, include oral,rectal, topical, nasal, pulmonary, ocular, intestinal, and parenteraladministration. Primary routes for parenteral administration includeintravenous, intramuscular, and subcutaneous administration. Secondaryroutes of administration include intraperitoneal, intra-arterial,intra-articular, intracardiac, intracisternal, intradermal,intralesional, intraocular, intrapleural, intrathecal, intrauterine, andintraventricular administration. The indication to be treated, alongwith the physical, chemical, and biological properties of the drug,dictate the type of formulation and the route of administration to beused, as well as whether local or systemic delivery would be preferred.

Pharmaceutical dosage forms of a compound of the invention may beprovided in an instant release, controlled release, sustained release,or target drug-delivery system. Commonly used dosage forms include, forexample, solutions and suspensions, (micro-) emulsions, ointments, gelsand patches, liposomes, tablets, dragees, soft or hard shell capsules,suppositories, ovules, implants, amorphous or crystalline powders,aerosols, and lyophilized formulations. Depending on route ofadministration used, special devices may be required for application oradministration of the drug, such as, for example, syringes and needles,inhalers, pumps, injection pens, applicators, or special flasks.Pharmaceutical dosage forms are often composed of the drug, anexcipient(s), and a container/closure system. One or multipleexcipients, also referred to as inactive ingredients, can be added to acompound of the invention to improve or facilitate manufacturing,stability, administration, and safety of the drug, and can provide ameans to achieve a desired drug release profile. Therefore, the type ofexcipient(s) to be added to the drug can depend on various factors, suchas, for example, the physical and chemical properties of the drug, theroute of administration, and the manufacturing procedure.Pharmaceutically acceptable excipients are available in the art, andinclude those listed in various pharmacopoeias. (See, e.g., USP, JP, EP,and BP, FDA web page (www.fda.gov), Inactive Ingredient Guide 1996, andHandbook of Pharmaceutical Additives, ed. Ash; Synapse InformationResources, Inc. 2002.)

Pharmaceutical dosage forms of a compound of the present invention maybe manufactured by any of the methods well-known in the art, such as,for example, by conventional mixing, sieving, dissolving, melting,granulating, dragee-making, tabletting, suspending, extruding,spray-drying, levigating, emulsifying, (nano/micro-) encapsulating,entrapping, or lyophilization processes. As noted above, thecompositions of the present invention can include one or morephysiologically acceptable inactive ingredients that facilitateprocessing of active molecules into preparations for pharmaceutical use.

Proper formulation is dependent upon the desired route ofadministration. For intravenous injection, for example, the compositionmay be formulated in aqueous solution, if necessary usingphysiologically compatible buffers, including, for example, phosphate,histidine, or citrate for adjustment of the formulation pH, and atonicity agent, such as, for example, sodium chloride or dextrose. Fortransmucosal or nasal administration, semisolid, liquid formulations, orpatches may be preferred, possibly containing penetration enhancers.Such penetrants are generally known in the art. For oral administration,the compounds can be formulated in liquid or solid dosage forms and asinstant or controlled/sustained release formulations. Suitable dosageforms for oral ingestion by a subject include tablets, pills, dragees,hard and soft shell capsules, liquids, gels, syrups, slurries,suspensions, and emulsions. The compounds may also be formulated inrectal compositions, such as suppositories or retention enemas, e.g.,containing conventional suppository bases such as cocoa butter or otherglycerides.

Solid oral dosage forms can be obtained using excipients, which mayinclude, fillers, disintegrants, binders (dry and wet), dissolutionretardants, lubricants, glidants, antiadherants, cationic exchangeresins, wetting agents, antioxidants, preservatives, coloring, andflavoring agents. These excipients can be of synthetic or naturalsource. Examples of such excipients include cellulose derivatives,citric acid, dicalcium phosphate, gelatine, magnesium carbonate,magnesium/sodium lauryl sulfate, mannitol, polyethylene glycol,polyvinyl pyrrolidone, silicates, silicium dioxide, sodium benzoate,sorbitol, starches, stearic acid or a salt thereof, sugars (i.e.dextrose, sucrose, lactose, etc.), talc, tragacanth mucilage, vegetableoils (hydrogenated), and waxes. Ethanol and water may serve asgranulation aides. In certain instances, coating of tablets with, forexample, a taste-masking film, a stomach acid resistant film, or arelease-retarding film is desirable. Natural and synthetic polymers, incombination with colorants, sugars, and organic solvents or water, areoften used to coat tablets, resulting in dragees. When a capsule ispreferred over a tablet, the drug powder, suspension, or solutionthereof can be delivered in a compatible hard or soft shell capsule.

In one embodiment, the compounds of the present invention can beadministered topically, such as through a skin patch, a semi-solid or aliquid formulation, for example a gel, a (micro-) emulsion, an ointment,a solution, a (nano/micro)-suspension, or a foam. The penetration of thedrug into the skin and underlying tissues can be regulated, for example,using penetration enhancers; the appropriate choice and combination oflipophilic, hydrophilic, and amphiphilic excipients, including water,organic solvents, waxes, oils, synthetic and natural polymers,surfactants, emulsifiers; by pH adjustment; and use of complexingagents. Other techniques, such as iontophoresis, may be used to regulateskin penetration of a compound of the invention. Transdermal or topicaladministration would be preferred, for example, in situations in whichlocal delivery with minimal systemic exposure is desired.

For administration by inhalation, or administration to the nose, thecompounds for use according to the present invention are convenientlydelivered in the form of a solution, suspension, emulsion, or semisolidaerosol from pressurized packs, or a nebuliser, usually with the use ofa propellant, e.g., halogenated carbons derived from methan and ethan,carbon dioxide, or any other suitable gas. For topical aerosols,hydrocarbons like butane, isobutene, and pentane are useful. In the caseof a pressurized aerosol, the appropriate dosage unit may be determinedby providing a valve to deliver a metered amount. Capsules andcartridges of, for example, gelatin, for use in an inhaler orinsufflator, may be formulated. These typically contain a powder mix ofthe compound and a suitable powder base such as lactose or starch.

Compositions formulated for parenteral administration by injection areusually sterile and, can be presented in unit dosage forms, e.g., inampoules, syringes, injection pens, or in multi-dose containers, thelatter usually containing a preservative. The compositions may take suchforms as suspensions, solutions, or emulsions in oily or aqueousvehicles, and may contain formulatory agents, such as buffers, tonicityagents, viscosity enhancing agents, surfactants, suspending anddispersing agents, antioxidants, biocompatible polymers, chelatingagents, and preservatives. Depending on the injection site, the vehiclemay contain water, a synthetic or vegetable oil, and/or organicco-solvents. In certain instances, such as with a lyophilized product ora concentrate, the parenteral formulation would be reconstituted ordiluted prior to administration. Depot formulations, providingcontrolled or sustained release of a compound of the invention, mayinclude injectable suspensions of nano/micro particles or nano/micro ornon-micronized crystals. Polymers such as poly(lactic acid),poly(glycolic acid), or copolymers thereof, can serve ascontrolled/sustained release matrices, in addition to others well knownin the art. Other depot delivery systems may be presented in form ofimplants and pumps requiring incision.

Suitable carriers for intravenous injection for the molecules of theinvention are well-known in the art and include water-based solutionscontaining a base, such as, for example, sodium hydroxide, to form anionized compound, sucrose or sodium chloride as a tonicity agent, forexample, the buffer contains phosphate or histidine. Co-solvents, suchas, for example, polyethylene glycols, may be added. These water-basedsystems are effective at dissolving compounds of the invention andproduce low toxicity upon systemic administration. The proportions ofthe components of a solution system may be varied considerably, withoutdestroying solubility and toxicity characteristics. Furthermore, theidentity of the components may be varied. For example, low-toxicitysurfactants, such as polysorbates or poloxamers, may be used, as canpolyethylene glycol or other co-solvents, biocompatible polymers such aspolyvinyl pyrrolidone may be added, and other sugars and polyols maysubstitute for dextrose.

For composition useful for the present methods of treatment, atherapeutically effective dose can be estimated initially using avariety of techniques well-known in the art. Initial doses used inanimal studies may be based on effective concentrations established incell culture assays. Dosage ranges appropriate for human subjects can bedetermined, for example, using data obtained from animal studies andcell culture assays.

A therapeutically effective dose or amount of a compound, agent, or drugof the present invention refers to an amount or dose of the compound,agent, or drug that results in amelioration of symptoms or aprolongation of survival in a subject. Toxicity and therapeutic efficacyof such molecules can be determined by standard pharmaceuticalprocedures in cell cultures or experimental animals, e.g., bydetermining the LD50 (the dose lethal to 50% of the population) and theED50 (the dose therapeutically effective in 50% of the population). Thedose ratio of toxic to therapeutic effects is the therapeutic index,which can be expressed as the ratio LD50/ED50. Agents that exhibit hightherapeutic indices are preferred.

The effective amount or therapeutically effective amount is the amountof the compound or pharmaceutical composition that will elicit thebiological or medical response of a tissue, system, animal, or humanthat is being sought by the researcher, veterinarian, medical doctor, orother clinician, e.g., regulation of glucose metabolism, decrease inelevated or increased blood glucose levels, treatment or prevention of adisorder associated with altered glucose metabolism, e.g., diabetes, etc

Dosages preferably fall within a range of circulating concentrationsthat includes the ED50 with little or no toxicity. Dosages may varywithin this range depending upon the dosage form employed and/or theroute of administration utilized. The exact formulation, route ofadministration, dosage, and dosage interval should be chosen accordingto methods known in the art, in view of the specifics of a subject'scondition.

Dosage amount and interval may be adjusted individually to provideplasma levels of the active moiety that are sufficient to achieve thedesired effects, e.g., regulation of glucose metabolism, decrease inblood glucose levels, etc., i.e., minimal effective concentration (MEC).The MEC will vary for each compound but can be estimated from, forexample, in vitro data and animal experiments. Dosages necessary toachieve the MEC will depend on individual characteristics and route ofadministration. In cases of local administration or selective uptake,the effective local concentration of the drug may not be related toplasma concentration.

The amount of agent or composition administered may be dependent on avariety of factors, including the sex, age, and weight of the subjectbeing treated, the severity of the affliction, the manner ofadministration, and the judgment of the prescribing physician.

The present compositions may, if desired, be presented in a pack ordispenser device containing one or more unit dosage forms containing theactive ingredient. Such a pack or device may, for example, comprisemetal or plastic foil, such as a blister pack, or glass and rubberstoppers such as in vials. The pack or dispenser device may beaccompanied by instructions for administration. Compositions comprisinga compound of the invention formulated in a compatible pharmaceuticalcarrier may also be prepared, placed in an appropriate container, andlabeled for treatment of an indicated condition.

These and other embodiments of the present invention will readily occurto those of ordinary skill in the art in view of the disclosure herein.

EXAMPLES

The invention will be further understood by reference to the followingexamples, which are intended to be purely exemplary of the invention.These examples are provided solely to illustrate the claimed invention.The present invention is not limited in scope by the exemplifiedembodiments, which are intended as illustrations of single aspects ofthe invention only. Any methods that are functionally equivalent arewithin the scope of the invention. Various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and accompanyingfigures. Such modifications are intended to fall within the scope of theappended claims.

Example 1 Overcoming Suppressive Effects of TNF-α on EPO Production

Hep3B cells were treated with various concentrations (0, 0.4, 2, 10ng/ml) of TNF-α in the absence or presence of compound A or compound Bfor 3 days. Secreted EPO levels were determined using a commerciallyavailable ELISA kit (R&D Systems, catalog no. DEP00). In the absence ofcompound, treatment of Hep3B cells with TNF-α reduced EPO production ina dose-dependent manner. Hep3B cells treated with various concentrationsof either compound A (FIG. 1A) or compound B (FIG. 1B) in the absence ofTNF-α showed a dose-dependent increase in EPO production. Addition ofeither compound in the presence of TNF-α greatly reduced the inhibitoryeffects of TNF-α on EPO production. Overcoming the suppressive effect ofTNF-α on EPO production by prolyl hydroxylase inhibition was observed inthe presence of low (e.g., 0.4 ng/ml) and high (e.g., 10 ng/ml)concentrations of TNF-α. Therefore, inhibitory effects of theinflammatory cytokine TNF-α on EPO production were overcome byinhibition of prolyl hydroxylase activity using compounds and methods ofthe present invention. These results suggested that compounds andmethods of the present invention are useful for increasing EPOproduction in the presence of the inflammatory cytokine TNF-α. Further,the methods and compounds of the present invention are useful toincrease EPO production and, therefore, to treat anemia in a subject,for example, wherein the subject has a disorder associated with TNF-

such as acute or chronic inflammation or other anemia of chronicdisease.

A series of experiments were performed to examine the effects ofcompounds of the present invention on EPO production following exposureof cells to the inflammatory cytokine TNF-α (i.e., in cells alreadyexposed to TNF-α). In these experiments, TNF-α signaling would thereforebe initiated prior to the addition of a prolyl hydroxylase inhibitor.Hep3B cells were treated with various concentrations (0, 0.4, 2, 10ng/ml) of TNF-α for 2 hours, after which various concentrations ofcompound A or compound B were added to the cultured cells. Secreted EPOlevels were determined as described above 3 days following compoundaddition.

As shown in FIGS. 2A and 2B, compound A and compound B overcame thesuppressive effects of TNF-α on EPO production following a 2-hourpre-treatment of Hep3B cells with TNF-α. This data indicated thatcompounds and methods of the present invention are useful for increasingEPO production in cells exposed to TNF-α. These results also suggestedthat treatment with compound of the present invention provides usefulmeans to increase EPO production and treat anemia in a subject in whichEPO production has been suppressed by TNF-α.

Addition of compounds of the present invention greatly reduced theinhibitory effects of TNF-α on EPO production. Therefore, compounds andmethods of the present invention are useful for treating or preventinganemia of associated with increased TNF-α, e.g., inflammatory disorders.

Example 2 Overcoming Suppressive Effects of IL-1 on EPO Production

Hep3B cells were treated with various concentrations (0, 0.4, 2, 10ng/ml) of IL-1β in the absence or presence of compound A or compound Bfor 3 days. Secreted EPO levels were determined using a commerciallyavailable ELISA kit (R&D Systems, catalog no. DEP00). In the absence ofcompound, treatment of Hep3B cells with IL-1β reduced EPO production ina dose-dependent manner. Hep3B cells treated with various concentrationsof either compound A (FIG. 3A) or compound B (FIG. 3B) in the absence ofIL-1β showed a dose-dependent increase in EPO production. Addition ofeither compound in the presence of IL-1β greatly reduced the inhibitoryeffects of IL-1β on EPO production. Overcoming the suppressive effectsof IL-1β on EPO production by prolyl hydroxylase inhibition was observedin the presence of low (e.g., 0.4 ng/ml) and high (e.g., 10 ng/ml)concentrations of IL-1β. Therefore, inhibitory effects of theinflammatory cytokine IL-1β on EPO production were overcome byinhibition of prolyl hydroxylase activity using compounds and methods ofthe present invention. These results suggested that compounds andmethods of the present invention are useful for increasing EPOproduction in the presence of the inflammatory cytokine IL-1β. Further,the methods and compounds of the present invention are useful toincrease EPO production and, therefore, to treat anemia in a subject,for example, wherein the subject has a disorder associated with IL-1

such as acute or chronic inflammation or other anemia of chronicdisease.

A series of experiments were performed to examine the effects ofcompounds of the present invention on EPO production following exposureof cells to the inflammatory IL-1β (i.e., in cells already exposed toIL-1β). In these experiments, IL-1β signaling would therefore beinitiated prior to the addition of a prolyl hydroxylase inhibitor. Hep3Bcells were treated with various concentrations (0, 0.4, 2, 10 ng/ml) ofIL-1β for 2 hours, after which various concentrations of compound A orcompound B were added to the cultured cells. Secreted EPO levels weredetermined as described above 3 days following compound addition.

As shown in FIGS. 4A and 4B, compound A and compound B overcame thesuppressive effects of IL-1β on EPO production following a 2-hourpre-treatment of Hep3B cells with IL-1β. This data indicated thatcompounds and methods of the present invention are useful for increasingEPO production in cells exposed to IL-1β. These results also suggestedthat treatment with compound of the present invention provides usefulmeans to increase EPO production and treat anemia in a subject in whichEPO production has been suppressed by IL-1β.

Addition of compounds of the present invention greatly reduced theinhibitory effects of IL-1β on EPO production. Therefore, compounds andmethods of the present invention are useful for treating or preventinganemia associated with IL-1β, e.g., inflammatory disorders.

Example 3 Inhibition of TNF-α Induced VCAM-1 Expression

Endothelial cell adhesiveness for lymphocytes occurs, in part, byendothelial cell expression of vascular cell adhesion molecule (VCAM)-1.VCAM-1 expression in endothelial cells is induced by variousinflammatory cytokines, such as TNF-α. To investigate the effect of HIFprolyl hydroxylase inhibition on TNF-α induced VCAM-1 expression, HUVEC(human umbilical vein endothelial cells) were stimulated with TNF-α inthe absence or presence of various concentrations of compound B orcompound C for 1 day. VCAM expression was then measured.

As shown in FIG. 5, TNF-α (1 ng/ml) induced VCAM-1 expression in HUVECcells. Addition of compound B or compound C to TNF-α stimulated cells,however, resulted in a does-dependent inhibition of TNF-α induced VCAM-1expression. This data indicated that methods and compounds of thepresent invention are effective at reducing VCAM-1 expression associatedwith the inflammatory cytokine TNF-α. The results further suggested thatcompounds and methods of the present invention are useful for inhibitingVCAM-1 expression associated with various inflammatory and autoimmunediseases, such as, for example, anemia of chronic disease.

Example 4 Inhibition of IL-1β Induced VCAM-1 Expression

VCAM-1 expression in endothelial cells is also induced by theinflammatory cytokine IL-1β. To investigate the effect of HIF prolylhydroxylase inhibition on IL-1β induced VCAM-1 expression, HUVEC (humanumbilical vein endothelial cells) were stimulated with IL-1β in theabsence or presence of various concentrations of compound B or compoundC for 1 day. VCAM expression was then measured.

IL-1β (1 ng/ml) induced VCAM-1 expression in HUVEC cells. Addition ofcompound B or compound C to IL-1β stimulated cells, however, resulted ina does-dependent inhibition of IL-1β induced VCAM-1 expression. (Datanot shown.) These results indicated that methods and compounds of thepresent invention are effective at reducing VCAM-1 expression associatedwith the inflammatory cytokine IL-1β. The results further suggested thatcompounds and methods of the present invention are useful for inhibitingVCAM-1 expression associated with various inflammatory and autoimmunediseases, such as, for example, anemia of chronic disease.

Example 5 Inhibition of TNF-α and IL-1β Induced VCAM-1 Expression onEndothelial Cells

HUVEC were treated with vehicle control or various concentrations (0,20, 40, 80 μM) of compound B or compound C for 24 hours. Cells werewashed and then stimulated with either 1 ng/ml TNF-α or 1 ng/ml IL-1βfor 4 hours. Cell surface VCAM-1 expression was then measured bycell-based ELISA.

As shown in FIG. 25, pretreatment with prolyl hydroxylase inhibitorsdecreased the induction of cell surface VCAM-1 expression induced by theinflammatory cytokines TNF-α and IL-1β. These results indicated thatcompounds and methods of the invention inhibited the inflammatoryfunction of TNF-α and IL-1β and inhibited the expression of cell surfaceadhesion molecules important for mediating heterocellular leukocyteadhesion. Inhibition of leukocyte adhesion by treatment with the presentcompounds provides an effective means for decreasing inflammatorycascades, thereby reducing inflammation and reducing the inflammatoryeffect of limiting EPO production and suppressing erythropoiesis.

Example 6 Inhibition of TNF-α Induced E-Selectin Expression

Endothelial E-selectin belongs to the selectin family of cellularadhesion molecules mediating the initial attachment of leukocytes tovascular endothelial cells in inflammatory events. IL-1, TNF-α, andlipopolysaccharides each induce the expression of E-selectin. (See,e.g., Bevilacqua et al. (1987) Proc Natl Acad Sci USA 84:9238-9242 andBevilacqua and van Furth (1993) J Leukoc Biol 54:363-378.) Toinvestigate the effect of HIF prolyl hydroxylase inhibition on TNF-αinduced E-selectin expression, HUVECs were stimulated with 1 ng/ml TNF-αin the absence or presence of various concentrations of compound B orcompound C for 1 day. E-selectin and VCAM expression were then measured.

As shown in FIGS. 24A and 24B, compound B and compound C showed adose-dependent inhibition of TNF-α induced VCAM and E-selectinexpression in HUVECs. Data in FIGS. 24A and 24B is presented as percentinhibition of VCAM and E-selectin expression observed in response tovarious concentrations of compound B (FIG. 24A) or compound C (FIG.24B). Greater than 60% inhibition of VCAM and E-selectin expression wasobserved in HUVEC treated with 50 μM compound B or compound C. This dataindicated that methods and compounds of the present invention areeffective at reducing VCAM and E-selectin expression in endothelialcells associated with the inflammatory cytokine TNF-α. The resultsfurther suggested that compounds and methods of the present inventionare useful for inhibiting VCAM and E-selectin expression associated withvarious inflammatory and autoimmune disorders, such as, for example,anemia of chronic disease. Additionally, inhibition of endothelial cellexpression of adhesion molecules, including VCAM and E-selectin, bymethods and compounds of the present invention provides means forreducing early events in vascular inflammation.

Example 7 Inhibition of IL-1β Induced E-Selectin Expression

To investigate the effect of HIF prolyl hydroxylase inhibition on IL-1βinduced E-selectin expression, HUVECs were stimulated with 1 ng/ml IL-1βin the absence or presence of various concentrations of compound B orcompound C for 1 day. E-selectin expression was then measured.

Compound B and compound C showed a dose-dependent inhibition of IL-1βinduced E-selectin expression in HUVECs. (Data not shown.) These resultsindicated that methods and compounds of the present invention areeffective at reducing E-selectin expression in endothelial cellsassociated with the inflammatory cytokine IL-1β. The results furthersuggested that compounds and methods of the present invention are usefulfor inhibiting E selectin expression associated with variousinflammatory and autoimmune disorders, such as, for example, anemia ofchronic disease. Additionally, inhibition of endothelial cell expressionof adhesion molecules, including VCAM and E-selectin, by methods andcompounds of the present invention provides means for reducing earlyevents in vascular inflammation.

Example 8 Inhibition of TNF-α, IL-1β, and IFN- Induced E-SelectinExpression

HUVEC were treated with vehicle control or various concentrations ofcompound B or compound C for 24 hours. Cells were washed and thenstimulated with either 1 ng/ml TNF-α, 1 ng/ml IL-1β, or a combination of1 ng/ml each of TNF-α, IL-1β, and IFN-

for 4 hours. Cell surface expression of E-selectin was measured bycell-based ELISA.

As shown in FIG. 26, pretreatment of HUVEC with compound B or compound Cinhibited the induction of cell surface E-selectin expression induced bythe inflammatory cytokines TNF-α or IL-1β. In addition, pretreatmentwith either compound decreased E-selectin expression in the presence ofthree inflammatory cytokines known to increase E-selectin expression(TNF-α, IL-β, and IFN-γ). These results indicated that the presentcompounds blocked the inflammatory function of TNF-α, IL-1β, and IFN-γon endothelial cells, as exemplified by inhibition of the expression ofcell surface adhesion molecules that mediate rolling of leukocytes onactivated endothelium. Since leukocyte adhesion to activated endotheliumvia E-selectin is an early step in perpetuating inflammatory cascades,inhibition of leukocyte rolling by inhibiting E-selectin expressionprovides a means for decreasing inflammatory cascades that further limitEPO production and suppress erythropoiesis.

Example 9 Synergistic Increase in EPO Production

Hep3B cells were treated with various concentrations (0, 0.1, 1, 10ng/ml) of IL-6 in the absence of presence of various concentrations (3μM, 10 μM, 30 μM) of compound A or compound B for 1 or 3 days. SecretedEPO levels were determined using a commercially available ELISA kit (R&DSystems, catalog no. DEP00). In the absence of compound, treatment ofHep3B cells with IL-6 had a minimal effect on EPO production. As shownin FIGS. 27A and 27B, Hep3B cells treated with IL-6 increased EPOexpression slightly above that in non-treated cells. Specifically, EPOlevels in control cells was approximately 20 mIU/ml, while that in cellstreated with 10 ng/ml IL-6 was approximately 50 mIU/ml.

Hep3B cells treated with compound A or compound B without IL-6 showedincreased EPO levels in a dose-dependent manner. Hep3B cells treatedwith compound A or compound B in the presence of IL-6, however, showed asignificant increase in EPO levels. (See FIGS. 27A and 27B.) The effectof compound treatment on EPO production in the presence of IL-6 wassynergistic. For example, Hep3B cells treated with 10 ng/ml IL-6 showedapproximately 50 mIU/ml EPO levels. Treatment of Hep3B cells with 10 mMcompound A or compound B in the absence of IL-6 resulted inapproximately 60 mIU/ml EPO and 220 mIU/ml, respectively. In thepresence of 10 ng/ml IL-6, compound A and compound B addition increasedEPO levels to approximately 270 mIU/ml and to greater than 400 mIU/ml,respectively. Therefore, compounds of the present invention actedsynergistically with IL-6 at inducing EPO expression in hepatocytes.

Example 10 Overcoming Cytokine-Induced Suppression of EPO ReceptorSignaling

The cell line TF-1 (human erythroleukemia; ATCC cat #CRL-2003) isstimulated to proliferate in response to EPO addition. In the presenceof various pro-inflammatory cytokines, the EPO-mediated increase in TF-1cell proliferation is attenuated. To determine the effects of prolylhydroxylase inhibition on TF-1 cell proliferation, TF-1 cells aretreated with the various concentrations of the pro-inflammatorycytokines IL-1β, TNF-α, or IFN-γ in the absence or presence of prolylhydroxylase inhibitors, and EPO-mediated cell proliferation is measuredas follows. Triplicate wells of cells cultured in 96-well microtiterplates are incubated with serum-free medium in the absence or presenceof EPO for 24 hours. During the final 4 hours of culture, 1 μCi oftritiated thymidine (³H-TdR; Amersham) is added to each well. Cellresponsiveness to EPO receptor signaling is determined by measuring cellproliferation. Cell proliferation is measured by quantitating the amountof ³H-TdR incorporated into cells, first by lysing the cells with waterand then capturing the DNA on nylon filters in a cell harvester.

Alternatively, single cell suspensions of splenic cells obtained fromphenylhydrazine-treated animals, which lead to prevalence of EPOresponsive progenitors in spleen, are used as the source of EPOresponsive cells. EPO-mediated proliferation is then assessed ex vivo asdescribed above.

TF-1 cells treated with EPO results in an increase in cellproliferation, as determined by an increase in tritiated thymidineincorporation. Addition of the pro-inflammatory cytokines IL-1β, TNF-α,or IFN-γ to EPO-treated TF-1 cells results in decreased responsivenessto EPO, leading to decreased cell proliferation. The effect of additionof the present compounds on the inhibitory effects of pro-inflammatorycytokines on EPO-mediated cell proliferation in TF-1 cells isdetermined. Increased cell proliferation, as measured by increasedtritiated thymidine incorporation, in TF-1 cells treated with EPO andpro-inflammatory cytokines indicates that compounds and methods of thepresent invention overcome the suppressive effects of pro-inflammatorycytokines on EPO-mediated increase in cell proliferation.

Example 11 Increasing Transferrin Receptor Expression

The effect of compounds of the invention on transferrin receptorexpression was examined as follows. Various cells (Hep3B, HepG2, HK-2)were incubated with compound A or compound B for 1 day. The cells werethen analyzed for transferrin receptor expression by FACS analysis usingCD71-PE antibody (Ancell, catalog no. 223-050). The results are shownbelow in Table 1.

TABLE 1 Cell Type Treatment Mean FL Hep3B DMSO 40.21 Compound A 40.89Compound B 42.43 HepG2 DMSO 49.59 Compound A 56.52 Compound B 53.53 HK-2DMSO 10.80 Compound A 12.20 Compound B 18.92

As shown above in Table 1, addition of various compounds of the presentinvention to cells increased expression of transferrin receptor.Inhibition of HIF prolyl hydroxylation using prolyl hydroxylaseinhibitors of the present invention increased transferrin receptorexpression in cells. Increased transferrin receptor expression usingprolyl hydroxylase inhibitors of the present invention was observed inliver cells (e.g., Hep3B, HepG2), kidney cells (e.g., HK-2), andlymphocytes (e.g., THP-1). Therefore, methods and compounds of thepresent invention are useful for increasing transferrin receptorexpression in various cell types. In addition, increased transferrinreceptor expression would result in increased transferrinreceptor-mediated endocytosis of ferric transferrin, thereby increasingiron transport, utilization, storage, and metabolism. Therefore, methodsand compounds of the present invention are useful for enhancingerythropoiesis by increasing iron transport, utilization, storage, andmetabolism.

Example 12 Increasing Transferrin Receptor Expression and Iron Uptake InVitro

The effect of compounds on iron uptake in cells is determined asfollows. Primary monocytes and macrophage, and monocyte and macrophagecell lines (e.g., THP-1), are treated for one, two, or three days withvarious concentrations of prolyl hydroxylase inhibitors. Cells are thenexamined for the presence of cell surface transferrin receptor usingfluorescent immunostaining and flow cytometry. Results showing thataddition of prolyl hydroxylase inhibitors increase cell surfacetransferrin receptor expression indicates effectiveness of prolylhydroxylase inhibition at increasing transferrin binding and, therefore,iron binding, to cells. A change in iron uptake by cells treated withprolyl hydroxylase inhibitors is determined as follows. Cells aretreated with compound in the presence of ⁵⁹Fe. Increased iron uptake bycells treated with prolyl hydroxylase inhibitors is determined bymeasuring cell-associated ⁵⁹Fe. An increase in cell-associated ⁵⁹Feindicates increased iron uptake in cells.

Example 13 Increasing Iron-Regulatory Protein-2 Levels and Activity

The regulation of iron uptake, storage, and utilization occur, in part,through the expression and activity of key proteins involved in ironmetabolism, including trans-acting proteins known as iron-regulatoryproteins (IRPs). IRP-1 and IRP-2 control mRNA stability and translationby binding to specific iron-responsive elements in various mRNAs ofproteins involved in iron metabolism, thereby affecting virtually allaspects of iron metabolism. Iron deficiency increases IRP activity,resulting in increased transferrin receptor expression and reducedferritin expression. Likewise, in the presence of iron, IRP activitydecreases, leading to decreased transferrin receptor expression andincreased ferritin expression.

To examine the effect of the present compounds on various aspects ofiron metabolism, the following experiment is performed. Mouse Hepa-1cells are treated with prolyl hydroxylase inhibitors for up to 48 hours.The cells are then harvested and cell lysates analyzed for IRP-2expression by immunoblotting using an antibody specific for IRP-2 (AlphaDiagnostic International, Inc., San Antonio Tex.). Results showingincreased levels of cytoplasmic IRP-2 following addition of compounddemonstrates that methods and compounds of the present invention areuseful for increasing IRP levels and therefore iron metabolism.

The effect of compounds of the invention on IRP-2 activity, as measuredby changes in ferritin and transferrin expression, is determined asfollows. Mouse RAW 264.1 macrophage cell line is treated with prolylhydroxylase inhibitors for up to 48 hours. Cells are then harvested andanalyzed for ferritin and transferrin protein expression byimmunoblotting (ADI, catalogue #IRP21-S). Decreased levels of ferritinexpression and increased levels of transferrin expression followingprolyl hydroxylase inhibition indicates that methods and compounds ofthe present invention are useful for stabilizing and increasing theactivity of IRP-2. Increased expression of IRP-2 decreases expression offerritin, which is responsible for long-term storage of iron, andincreases expression of transferrin and transferrin receptor,facilitating iron uptake, transport, and utilization, thus enhancingerythropoiesis. By increasing expression and activity of IRP-2, methodsand compounds of the present invention are useful for decreasingexpression of ferritin and associated long-term storage of iron, andincreasing expression of transferrin and transferrin receptor.Therefore, methods and compounds of the present invention are useful forincreasing iron uptake, transport, and utilization, and are thus usefulfor enhancing erythropoiesis.

Example 14 Enhancing Iron Utilization

Rats are administered either vehicle control or HIF prolyl hydroxylaseinhibitors prior to intravenous injection with ⁵⁹Fe-radiolabeled ferrouscitrate (Amersham). Serial samples of blood are drawn from the tail veinand total free plasma and erythrocyte-associated radioactivity ismeasured in a scintillation counter to detect iron transport andincorporation into erythrocyte heme and hemoglobin synthesis. Increasein erythrocyte-associated ⁵⁹Fe indicates that the present compounds areuseful for enhancing iron utilization necessary for heme synthesis,hemoglobin production, and erythropoiesis.

Example 15 Enhanced Expression of Erythropoiesis Genes In Vitro

Hep3B cells (ATCC No. HB-8064) were grown in DMEM containing 8% fetalbovine serum. Hep3B cells were seeded into 6-well culture dishes at˜500,000 cells per well. After 8 hours, the media was changed to DMEMcontaining 0.5% fetal bovine serum and the cells were incubated for anadditional 16 hours. Compound B or compound D was added to the cells (25μM final concentration) and the cells were incubated for various times.Control cells (no compound treatment, addition of DMSO alone) wereharvested at 0, 6 and 48 hours. Harvested cells were assessed for cellviability (GUAVA), or added to RNA extraction buffer (RNeasy, Qiagen)and stored at −20° C. for subsequent RNA purification. Replicatemicroarrays were generated using RNA isolated from replicate experimentsconducted on different days. Total RNA was isolated from cells using theRNeasy kit (Qiagen).

RNA was precipitated in 0.3 M sodium acetate (pH 5.2), 50 ng/mlglycogen, and 2.5 volumes of ethanol for one hour at −20° C. Sampleswere centrifuged and pellets were washed with cold 80% ethanol, dried,and resuspend in water. Double stranded cDNA was synthesized using aT7-(dT)24 first strand primer (Affymetrix, Inc., Santa Clara Calif.) andthe SUPERSCRIPT CHOICE system (Invitrogen) according to themanufacturer's instructions. The final cDNA was extracted with an equalvolume of 25:24:1 phenol:chloroform:isoamyl alcohol using a PHASE LOCKGEL insert (Brinkman, Inc., Westbury N.Y.). The aqueous phase wascollected and cDNA was precipitated using 0.5 volumes of 7.5 M ammoniumacetate and 2.5 volumes of ethanol. Alternatively, cDNA was purifiedusing the GENECHIP sample cleanup module (Affymetrix) according to themanufacturer's instructions.

Biotin-labeled cRNA was synthesized from the cDNA in an in vitrotranslation (IVT) reaction using a BIOARRAY HighYield RNA transcriptlabeling kit (Enzo Diagnostics, Inc., Farmingdale N.Y.) according to themanufacturer's instructions. Final labeled product was purified andfragmented using the GENECHIP sample cleanup module (Affymetrix)according to the manufacturer's instructions.

Hybridization cocktail was prepared by bringing 5 μg probe to 100 μl in1× hybridization buffer (100 mM MES, 1 M [Na⁺], 20 mM EDTA, 0.01% Tween20), 100 μg/ml herring sperm DNA, 500 μg/ml acetylated BSA, 0.03 nMcontrol oligo B2 (Affymetrix), and 1× GENECHIP eukaryotic hybridizationcontrol (Affymetrix). The cocktail was sequentially incubated at 99° C.for 5 minutes and 45° C. for 5 minutes, and then centrifuged for 5minutes. The Human Genome U133A array (Affymetrix) was brought to roomtemperature and then prehybridized with 1× hybridization buffer at 45°C. for 10 minutes with rotation. The buffer was then replaced with 80 μlhybridization cocktail and the array was hybridized for 16 hours at 45°C. at 60 rpm with counter balance. Following hybridization, arrays werewashed once with 6×SSPE, 0.1% Tween 20, and then washed and stainedusing R-phycoerythrin-conjugated streptavidin (Molecular Probes, EugeneOreg.), goat anti-streptavidin antibody (Vector Laboratories, BurlingameCalif.), and a GENECHIP Fluidics Station 400 instrument (Affymetrix)according to the manufacturer's micro_(—)1v1 protocol (Affymetrix).Arrays were analyzed using a GENEARRAY scanner (Affymetrix) andMicroarray Suite software (Affymetrix).

The Human Genome U133A array (Affymetrix) represents all sequences inthe Human Unigene database build 133 (National Center for BiotechnologyInformation, Bethesda Md.), including approximately 14,500well-characterized human genes.

RNA quality was monitored by capillary electrophoresis (AgilentBioanalyzer). Hybridization cocktails were prepared as described(Affymetrix), and hybridized to Affymetrix human U133A arrays containing22,283 probe sets. Array performance was analyzed with AffymetrixMicroArray Suite (MAS) software and individual probe sets were assigned“present”, “marginal, and “absent” calls according to software defaults.Statistical analyses and filtered probe set lists were prepared usingGeneSpring software (Silicon Genetics). Cutoffs for “expressed” probesets used a combination of Affymetrix “P” calls and absolute expressionvalues derived from Genespring's intrinsic data error model. Data wasnormalized to averaged control samples.

As shown in Table 2 below, expression of genes (fold-increase in mRNAlevels above control) encoding erythropoietic proteins was increased inHep3B cells treated with compound of the present invention. (Twoceruloplasmin data points for each condition are presented below inTable 2.) Specifically, ceruloplasmin and transferrin receptor 2 geneexpression were increased in Hep3B cells treated with various compoundsof the present invention.

TABLE 2 Ceruloplasmin Transferrin Receptor Compound Time (CP) (TFR2) D 6 hr  2.06/2.387 Not Determined B  1 hr 1.142/0.946 0.575 B  3 hr1.123/0.955 0.558 B  6 hr 1.555/1.103 0.822 B 12 hr 2.366/2.507 1.253 B24 hr 5.136/4.909 2.522 B 48 hr  5.82/4.678 4.169

Example 16 Animal Dosing

Animals used in the following examples include Swiss Webster male mice(30-32 g), Sprague Dawley male rats (200-350 g) and Lewis female ratesobtained from Simonsen, Inc. (Gilroy Calif.), Charles River (Hollister,Calif.), or Harlan. Animals were maintained using standard procedures,and food and water were available to the animals ad libitum. Duringtreatment, animals were monitored for changes in body weight and signsof overt toxicity and mortality.

Compounds were generally administered orally by gavage or IVadministration. Animals treated by oral gavage received a 4-10 ml/kgvolume of either 0.5% carboxymethyl cellulose (CMC; Sigma-Aldrich, St.Louis Mo.) with or without 0.1% Polysorbate 80 (0 mg/kg/day) or varyingdoses of a compound of the present invention (e.g., a HIF prolylhydroxylase inhibitor) in 0.5% CMC, with or without 0.1% Polysorbate 80,using various dosing regimens. Blood samples were collected atappropriate intervals during treatment from, e.g., tail vein (rats), orabdominal vein or cardiocentesis (mice or rats). Generally, animals wereanesthetized with isoflurane and blood samples were collected intoMICROTAINER serum separator tubes (Becton-Dickinson, Franklin LakesN.J.). For measurement of serum components, the tubes were incubated atroom temperature for 30 minutes, and then centrifuged at 8,000 rpm at 4°C. for 10 minutes. The serum fraction was then processed and analyzedfor the presence of specific components, e.g., serum iron (assayperformed by Quality Clinical Labs, Mountain View, Calif.). Fordetermination of hematocrit, blood samples were collected intoMICROTAINER EDTA-2K tubes (Becton-Dickinson); EDTA-blood was then drawninto 75 mm×1.1-1.2 mm I.D. capillary tubes (Chase Scientific Glass,Inc., Rockwood Tenn.) to approximately ¾ length, one end of the tube wassealed with CRITOSEAL sealant (Sherwood Medical Company), and the tubeswere centrifuged in a J-503M MICROHEMATOCRIT centrifuge (JorgensenLaboratories, Inc., Loveland Colo.) at 12,000 rpm for 5 minutes.Hematocrit was read against a reader card. When indicated, completeblood count (CBC) analysis, including blood hemoglobin level,reticulocyte number, and hematocrit, was performed by Quality ClinicalLabs (Mountain View, Calif.).

At the end of each study, animals were euthanized, e.g. byexsanguinations under general anesthesia or by CO₂ asphyxiation, andorgan and tissue samples were collected. Tissues were either fixed inneutral buffered formalin or stored frozen at −70° C. Tissues forgenomic analysis were placed in RNAlater.

Example 17 Increased Expression of Genes Encoding Iron-ProcessingProteins In Vivo

Swiss Webster male mice were treated as described above with a singledose of 0.5% CMC (Sigma-Aldrich) (0 mg/kg) or 100 mg/kg compound A. At4, 8, 16, 24, 48, or 72 hours post-administration, animals wereanesthetized, sacrificed, and tissue samples of kidney, liver, brain,lung, and heart were isolated and stored in RNALATER solution (Ambion)at −80° C. Alternatively, animals were treated to 4 consecutive dailydoses of 0.5% CMC (0 mg/kg/day), 7.5 mg/ml compound A in 0.5% CMC (30mg/kg/day), or 25 mg/ml compound A in 0.5% CMC (100 mg/kg/day). Fourhours after administration of the final dose, animals were anesthetized,sacrificed, and approximately 150 mg of liver and each kidney wereisolated and stored in RNALATER solution (Ambion) at −20° C.

RNA isolation was carried out using the following protocol. A section ofeach organ was diced, 875 μl of RLT buffer (RNEASY kit; Qiagen Inc.,Valencia Calif.) was added, and the pieces were homogenized for about 20seconds using a rotor-stator POLYTRON homogenizer (Kinematica, Inc.,Cincinnati Ohio). The homogenate was micro-centrifuged for 3 minutes topellet insoluble material, the supernatant was transferred to a new tubeand RNA was isolated using an RNEASY kit (Qiagen) according to themanufacturer's instructions. The RNA was eluted into 80 μL of water andquantitated with RIBOGREEN reagent (Molecular Probes, Eugene Oreg.). Theabsorbance at 260 and 280 nm was measured to determine RNA purity andconcentration.

Alternatively, tissue samples were diced and homogenized in TRIZOLreagent (Invitrogen Life Technologies, Carlsbad Calif.) using arotor-stator POLYTRON homogenizer (Kinematica). Homogenates were broughtto room temperature, 0.2 volumes chloroform was added, and samples weremixed vigorously. Mixtures were incubated at room temperature forseveral minutes and then were centrifuged at 12,000 g for 15 min at 4°C. The aqueous phase was collected and 0.5 volumes of isopropanol wereadded. Samples were mixed, incubated at room temperature for 10 minutes,and centrifuged for 10 min at 12,000 g at 4° C. The supernatant wasremoved and the pellet was washed with 75% EtOH and centrifuged at 7,500g for 5 min at 4° C. The absorbance at 260 and 280 nm was measured todetermine RNA purity and concentration.

RNA was precipitated in 0.3 M sodium acetate (pH 5.2), 50 ng/mlglycogen, and 2.5 volumes of ethanol for one hour at −20° C. Sampleswere centrifuged and pellets were washed with cold 80% ethanol, dried,and resuspend in water. Double stranded cDNA was synthesized using aT7-(dT)24 first strand primer (Affymetrix, Inc., Santa Clara Calif.) andthe SUPERSCRIPT CHOICE system (Invitrogen) according to themanufacturer's instructions. The final cDNA was extracted with an equalvolume of 25:24:1 phenol:chloroform:isoamyl alcohol using a PHASE LOCKGEL insert (Brinkman, Inc., Westbury N.Y.). The aqueous phase wascollected and cDNA was precipitated using 0.5 volumes of 7.5 M ammoniumacetate and 2.5 volumes of ethanol. Alternatively, cDNA was purifiedusing the GENECHIP sample cleanup module (Affymetrix) according to themanufacturer's instructions.

Biotin-labeled cRNA was synthesized from the cDNA in an in vitrotranslation (IVT) reaction using a BIOARRAY HighYield RNA transcriptlabeling kit (Enzo Diagnostics, Inc., Farmingdale N.Y.) according to themanufacturer's instructions. Final labeled product was purified andfragmented using the GENECHIP sample cleanup module (Affymetrix)according to the manufacturer's instructions.

Hybridization cocktail was prepared by bringing 5 μg probe to 100 μl in1× hybridization buffer (100 mM MES, 1 M [Na⁺], 20 mM EDTA, 0.01% Tween20), 100 μg/ml herring sperm DNA, 500 μg/ml acetylated BSA, 0.03 nMcontrol oligo B2 (Affymetrix); and 1× GENECHIP eukaryotic hybridizationcontrol (Affymetrix). The cocktail was sequentially incubated at 99° C.for 5 minutes and 45° C. for 5 minutes, and then centrifuged for 5minutes. The Murine genome MOE430Aplus2 array (Affymetrix) was broughtto room temperature and then prehybridized with 1× hybridization bufferat 45° C. for 10 minutes with rotation. The buffer was then replacedwith 80 μl hybridization cocktail and the array was hybridized for 16hours at 45° C. at 60 rpm with counter balance. Following hybridization,arrays were washed once with 6×SSPE, 0.1% Tween 20, and then washed andstained using R-phycoerythrin-conjugated streptavidin (Molecular Probes,Eugene Oreg.), goat anti-streptavidin antibody (Vector Laboratories,Burlingame Calif.), and a GENECHIP Fluidics Station 400 instrument(Affymetrix) according to the manufacturer's EukGE-WS2v4 protocol(Affymetrix). Arrays were analyzed using a GENEARRAY scanner(Affymetrix) and Microarray Suite software (Affymetrix).

The Murine Genome MOE430Aplus2 array (Affymetrix) represents allsequences in the Murine UniGene database build 107 (National Center forBiotechnology Information, Bethesda Md.), including approximately 14,000well-characterized mouse genes.

Table 3 below shows ceruloplasmin mRNA expression in mouse kidneyfollowing administration of compound A. Data was normalized to theaverage value of that observed in control non-treated animals.

TABLE 3 Ceruloplasmin Condition (relative mRNA levels) Untreated 0.81CMC control 1.26 Compound A - 4 hours 1.16 Compound A - 8 hours 1.39Compound A - 16 hours 1.22 Compound A - 24 hours 2.45 Compound A - 48hours 1.44 Compound A - 72 hours 2.10

Data shown in Table 3 above demonstrated that methods and compounds ofthe present invention are useful for increasing ceruloplasmin geneexpression. Ceruloplasmin, also known as a ferroxidase-1, convertsreduced iron released from storage sites (such as ferritin) to theoxidized form. Oxidized iron is able to bind to its plasma transportprotein, transferrin. Ceruloplasmin deficiencies are associated withaccumulation of iron in liver and other tissues. Evidence indicates thatceruloplasmin promotes efflux of iron from the liver and promotes influxof iron into iron-deficient cells. (See, e.g., Tran et al. (2002) J Nutr132:351-356.)

Table 4 below shows hepcidin mRNA expression in mouse liver followingadministration of compound A. Data was normalized to that observed incontrol non-treated animals.

TABLE 4 Hepcidin Condition/Animal Study Time (relative mRNA levels)Control — 1.0 I - multi high dose — 0.275 II - multi high dose — 0.703II - multi low dose — 0.129 III 4 hour 0.672 III 8 hour 0.305 III 16hour  0.119

As shown above in Table 4, administration of compound A resulted inreduced expression of hepcidin mRNA in mouse liver. Decreased hepcidinexpression is associated with increased iron release fromreticuloendothelial cells and increased intestinal iron absorption.Therefore, methods and compounds of the present invention are useful fordecreasing hepcidin expression and increasing intestinal ironabsorption.

FIG. 6A shows relative expression levels of the transferrin receptor(gray bars) in kidney, and the gut duodenal iron transporter NRAMP2(natural-resistance-associated macrophage protein 2) (also known asSlc11a2 (solute carrier family 11, proton-coupled divalent metal iontransporter, member 2), alternatively called DCT1 (divalent cationtransporter 1), DMT1 (divalent metal transporter 1)) (black bars).

In another experiment, mRNA was isolated from small intestine harvested4 hours following IV administration of 60 mg/kg compound A, compound B,and compound C to mice. Probes were prepared from each of two animalsfrom 5 treatment groups, and hybridized to Affymetrix mouse MOE430Aplus2microarrays (one animal per array). Statistical comparisons of dataobtained from arrays from treated versus non-treated animals wasperformed. FIG. 6B shows relative expression levels of NRAMP2 mRNA insmall intestine in animals treated with compound A, compound B, andcompound C. Expression levels are shown as fold-induction over control,untreated animals for each expressed gene. The results from theseexperiments indicated that methods and compounds of the presentinvention are useful for increasing expression of NRAMP2 in intestine.These results further suggested that methods and compounds of thepresent invention are useful for increasing iron absorption, therebyincreasing iron availability for heme synthesis, hemoglobin synthesis,red blood cell production, and erythropoiesis.

FIG. 6C shows the fold-induction of 5-aminolevulinate synthase (ALAS-2)expression in treated animals as compared to vehicle control. The datashowed that treatment of normal animals with prolyl hydroxylaseinhibitors resulted in increased expression of genes involved in ironmetabolism, including genes involved in iron absorption from the gut andiron transport in the periphery via transferrin receptors. Expression ofthese genes returned to baseline (control) levels 16 hours after dosing.The data also showed coordinate expression of ALAS-2, the first enzymein the heme synthetic pathway and rate-limiting enzyme for hemesynthesis, in the indicated tissues after prolyl hydroxylase inhibitortreatment. Together these results showed compounds of the presentinvention coordinated increases in expression of genes encoding proteinsinvolved in promoting erythropoiesis, including iron absorption, irontransport, and heme synthesis.

Alternatively, flow cytometry analysis is used to measure macrophagecell surface marker CD11c and transferrin receptor levels in doubleimmunostained peripheral blood mononuclear cells. Activity is shown forcompound treatment by detecting increased macrophage transferrinreceptor expression. Also, plasma can be collected and tested for levelsof transferrin using a commercially available ELISA kit (see, e.g.,KomaBiotech, Korea).

Example 18 Enhanced Erythropoiesis In Vivo

The effect of administration of the present compounds on erythropoiesisis determined as follows. Normal mice are made anemic and maintained inan anemic state by chronic administration of TNF-α, a regimen known toinhibit erythropoiesis due to lack of EPO production and signaling inresponse to TNF-α. After inducing anemia over a one- to four-weekperiod, animals are administered prolyl hydroxylase inhibitors. Tissuesare examined for BFU-E and CFU-E production, and blood samples areanalyzed for composition. Results showing increases in the numbers ofBFU-E and CFU-E in the marrow, spleen, and periphery, and/or increasesserum hemoglobin, reticulocytes, and hematocrit in animals treated withPHIs demonstrate efficacy.

Another experimental animal model is useful for examining the effect ofadministration of prolyl hydroxylase inhibitors on erythropoiesis. Inthis model, transgenic mice develop anemia of chronic disease as aresult of constitutively over expressing TNF-α. Following onset ofanemia in these mice, prolyl hydroxylase inhibitors are administered forvarious periods of time and using various dosing strategies. Tissue andblood samples are then collected and analyzed. As described above,results showing increases in the numbers of BFU-E and CFU-E in themarrow, spleen and periphery, and/or increased serum hemoglobin,reticulocytes and hematocrit, effectively demonstrate that anemiaassociated with TNF-α overproduction in transgenic animals is treated byadministration of prolyl hydroxylase inhibitors using methods andcompounds of the present invention.

Example 19 Increasing Serum Iron Levels

Male and female rats were treated twice weekly (Monday and Thursday)with various concentrations (0, 20, 60, or 150 mg/kg) of compound A for93 days. Total serum iron levels were determined.

TABLE 5 Serum Iron (□g/dL) Serum Iron (□g/dL) Dose Male Rats (Mean +/−SD) Female Rats (Mean +/− SD)  0 mg/kg 158 +/− 37 342 +/− 91  20 mg/kg198 +/− 64 505 +/− 41* 60 mg/kg  357 +/− 111* 445 +/− 46* 150 mg/kg  307 +/− 142* 399 +/− 117

As shown in Table 5, administration of compound A increased serum ironlevels in both male and female rats. (Data in Table 5 is presented asserum iron levels+/−standard deviation. * indicates a significantdifference in serum iron levels from non-treated animals.) These resultsindicated that methods and compounds of the present invention are usefulfor increasing serum iron levels, thereby useful for treating disordersassociated with iron deficiency.

Example 20 Efficacy in Animal Model of Anemia of ChronicDisease/Impaired Erythropoiesis/Impaired Iron Metabolism

Anemia of chronic disease (ACD) is associated with various inflammatoryconditions, including arthritis, neoplastic disease, and other disordersassociated with chronic inflammation. A rat model of ACD was used toexamine the effects of HIF stabilization using methods and compounds ofthe present invention on treating anemia associated with chronicdisease. In this animal model, ACD is induced in rats bypeptidoglycan-polysaccharide polymers. (See, e.g., Sartor et al. (1989)Infection and Immunity 57:1177-1185.) In this model, animals developsevere, acute anemia in the initial stages, followed by moderatelysevere chronic microcytic anemia in later stages.

Animal Model of ACD—Experimental Series 1:

Female Lewis rats of approximately 160 grams were challenged with PG-PS10S (Lee Laboratories, 15 μg/gm body weight, intra-peritoneal). PG-PS10S contains purified peptidoglycan-polysaccharide polymers isolatedfrom the cell wall of Streptococcus pyogenes, Group A, D58 strain.Arthritis and anemia were allowed to develop for 35 days. On day 35,blood samples (approximately 400 μl) were taken from the tail vein undergeneral anesthesia (Isoflurane) for CBC and reticulocyte counts(performed by Quality Clinical Labs). Animals with a spun hematocritlevel at or above 45% were considered non-anemic and were removed fromthe study.

On day 35 following PG-PS injection, anemic animals received vehiclealone or were treated with compound A (60 mg/kg, PO) for two consecutivedays per week for two weeks. Automated complete blood counts (CBC) weremeasured on day 35 (see above), 39, 42, and 49; serum iron levels weremeasured on day 49.

Reticulocyte Count

As shown in FIG. 7, administration of compound A to anemic animalsincreased reticulocyte count at day 39 (i.e., 5 days after initiation ofcompound dosing). Reticulocytes levels were approximately 2% and 4% ofred cells in control (non-anemic) and anemic (PG-PS treated) animals,respectively. Reticulocyte levels in treated animals, however, wereapproximately 10% of red cell counts. Compound A treatment increasedreticulocyte count in anemic animals. Therefore, compound A stimulatederythropoiesis in a rat animal model of ACD.

Hematocrit

Hematocrit levels were increased in anemic animals treated with compoundA. Hematocrit levels (measured by Baker 9000 at Quality Clinical Labs)in anemic animals (PG-PS treated) were less than 35%, compared to 41% incontrol non-anemic animals. (See FIG. 8.) Administration of compound Ato anemic animals increased hematocrit levels to approximately 37% asearly as 5 days after initiation of compound treatment. Following asecond dosing of compound A, hematocrit levels increased toapproximately 40%, comparable to hematocrit levels observed in controlnon-anemic animals. Compound A increased hematocrit in anemic animalsusing a rat model of ACD. Therefore, methods and compounds of thepresent invention are useful for increasing hematocrit and treatinganemia of chronic disease.

Hemoglobin

Compound A administration also increased hemoglobin levels in anemicanimals. As shown in FIG. 9, at day 35, control non-anemic animals hadhemoglobin levels of approximately 15 gm/dL, whereas hemoglobin levelsin PG-PS treated animals (i.e., anemic animals) were approximately 13gm/dL. As shown in FIG. 9, compound A increased hemoglobin levels inanemic animals as early as 5 days (day 39) following compoundadministration. Hemoglobin levels remained elevated at day 49, reachinga level comparable to control non-anemic animals, indicating compound ofthe present invention restored normal hemoglobin levels in anemicanimals. These results showed compound A increased hemoglobin in anemicanimals using a rat model of ACD. Therefore, methods and compounds ofthe present invention are useful for increasing hemoglobin and treatinganemia of chronic disease.

Red Blood Cell Count

Administration of compound A increased red blood cell count in anemicanimals. As shown in FIG. 10, red blood cell counts were increased inanemic animals treated with compound A compared to non-treated anemicanimals as early as 5 days after initiation of compound administration(i.e., day 39 in FIG. 10). Compound A increased red blood cell count inanemic animals using a rat model of ACD. Therefore, methods andcompounds of the present invention are useful for increasing red bloodcell count and treating anemia of chronic disease.

Mean Corpuscular Volume

Anemic animals showed reduced mean corpuscular volume compared tonon-anemic control animals. (See FIG. 11.) Anemic animals treated withcompound A showed increased mean corpuscular volume as early as 5 daysafter treatment (day 39 in FIG. 11) compared to non-treated anemicanimals. Mean corpuscular volume in treated animals remained elevatedcompared to non-treated anemic animals over the duration of theexperiment. These results showed that compound A improved (i.e.,reduced) the level of microcytosis (i.e., microcythemia, the presence ofmany microcytes, abnormally small red blood cells associated withvarious forms of anemia). Therefore methods and compounds of the presentinvention improve/reduce microcytosis in anemia of chronic disease.

Mean Corpuscular Hemoglobin

Anemic animals also showed reduced mean corpuscular hemoglobin levels.As shown in FIG. 12, treatment of anemic animals with compound Aincreased mean corpuscular hemoglobin levels above those observed innon-treated anemic animals. These results indicated that methods andcompounds of the present invention are useful to increase meancorpuscular hemoglobin levels.

Animal Model of ACD—Experimental Series 2:

Female Lewis rats (approximately 150-200 gm) were injected with PG-PS(intra-peritoneal). Arthritis and anemia were allowed to develop for 28days. Animals were administered compound A by oral gavage twice a week(Monday and Thursday) for six weeks, corresponding to days 28, 31, 35,38, 42, 45, 49, 52, 56, 59, 63, 66, and 70 from PG-PS injection.

Whole blood was collected via the tail vein for CBC analysis on days 28,42, 56, and 70. In addition, serum was collected on day 70 for ironbinding analysis. CBC and iron binding analysis were performed byQuality Clinical Labs (Mountain View, Calif.).

Hematocrit

Hematocrit levels were reduced in animals 28 days following challengewith PG-PS. FIG. 13 shows animals injected with PG-PS were anemic,having a hematocrit of 85% of that in non-challenged (i.e., non-anemic)animals. (Week 0 in FIG. 13 corresponds to day 28 in this experimentalprotocol.) Non-challenged (i.e., non-anemic) animals treated withcompound A (40 mg/kg) showed an increase in hematocrit levels over time,to greater than 110% of that in non-challenged non-treated animals. Asshown in FIG. 13, administration of compound A to anemic animalsresulted in increased hematocrit levels.

Hemoglobin

Compound A administration increased hemoglobin levels in both anemic andnon-anemic animals. As shown in FIG. 14, hemoglobin levels in non-anemicanimals treated with compound A (40 mg/kg) increased to approximately110% of that in non-treated control animals. (Week 0 in FIG. 14corresponds to day 28 in this experimental protocol.) In anemic animals,hemoglobin levels increased upon administration twice weekly of 10mg/kg, 20 mg/kg, or 40 mg/kg compound A. Hematocrit levels continued toincrease for at least 4 weeks.

Red Blood Cell Count

Anemic animals had lower red blood cell counts than non-anemic animals.Specifically, red blood cell counts in anemic animals were less than 90%of that observed in non-anemic animals at 28 days following PG-PSinjection. As shown in FIG. 15, red blood cell counts were increased inanemic animals treated with compound A compared to non-treated animals.(Week 0 in FIG. 15 corresponds to day 28 in this experimental protocol.)Increased red blood cell counts were observed at 2 weeks followingadministration of compound, and continued to increase over the 6 weekexperimental period.

Mean Corpuscular Volume

Anemic animals showed reduced mean corpuscular volume compared tonon-anemic (no challenge) animals. As shown in FIG. 16, mean corpuscularvolume in animals treated with PG-PS continued to decrease over time,indicating the effects of anemia of chronic disease resulted inmicrocytic anemia (characterized, in part, by lower red cell number andsmaller red cells), and the inability to produce hemoglobin due to ironstores being unavailable for utilization. (Week 0 in FIG. 16 correspondsto day 28 in this experimental protocol.) Administration of compound Ato anemic animals resulted in reduction of the decrease in meancorpuscular volume. Therefore, inhibition of prolyl hydroxylase usingcompounds and methods of the present invention was effective at reducingthe decrease in mean corpuscular volume associated with anemia ofchronic disease and anemia associated with iron deficiency, restoringmean corpuscular volume, maintaining mean corpuscular volume, etc. Thisdata further indicated that methods and compounds of the presentinvention are useful for increasing iron availability from storage foruse in hemoglobin production.

Mean Corpuscular Hemoglobin

Anemic animals had decreased mean corpuscular hemoglobin levels comparedto control animals, indicating anemia of chronic disease affectedhemoglobin production. As shown in FIG. 17, anemic animals administeredcompound A showed a reduction in the decrease in mean corpuscularhemoglobin levels over time. (Week 0 in FIG. 17 corresponds to day 28 inthis experimental protocol.)

Iron Status—Serum Iron and Transferrin Saturation

Patients with anemia of chronic disease are clinically characterized byreduced plasma iron concentrations and transferrin saturation. Theeffect of the present compounds on serum iron and transferrin saturationin normal and anemic animals was determined. Using an animal model ofanemia of chronic disease, anemia was induced in rats by IP injection ofpeptidoglycan-polysaccharide polymers, as described above. Arthritis andanemia were allowed to develop for 28 days. Animals were then treatedwith various concentrations of compound A, twice weekly, for 6 weeks.Serum iron levels and transferrin saturation were determined by QualityClinical Labs.

As shown in FIG. 18A, anemic animals (PG-PS) had-lower serum iron levelscompared to non-anemic animals (sham). Administration of compound Aresulted in increased serum iron levels in both anemic (PG-PS) andnon-anemic control (sham) animals. Animals treated with compound A hadincreased transferrin saturation compared to non-treated non-anemicanimals and to non-treated anemic animals. (See FIG. 18B.) These resultsindicated that methods and compounds of the present invention are usefulfor increasing serum iron levels and percent transferrin saturation.

Iron Absorption

At week 6 following administration of compound A in anemic animals (40mg/kg, twice a week), microarray analysis was performed to examineexpression of genes encoding proteins involved with iron transport andabsorption in intestine. Microarray analysis was performed using methodsdescribed above, using The Rat Genome 230A array (Affymetrix), whichrepresents all sequence in the Rat Unigene database build 99 (NationalCenter for Biotechnology Information, Bethesda, Md.), includingapproximately 4,699 well-characterized rat genes and approximately10,467 EST sequences and approximately 700 non-EST sequences.

As shown in FIG. 19, administration of compound A to control animalsincreased intestinal expression of mRNA for NRAMP2 (open bars) andsproutin (solid bars). Non-treated anemic animals (PG-PS) had reducedmRNA expression levels for both NRAMP2 and sproutin. These resultsindicated that anemia of chronic disease is associated with reducedexpression of proteins involved in iron absorption. Anemic animalstreated with compound A, however, showed increased expression of bothNRAMP2 and sproutin in intestine (FIG. 19). These results indicated thatmethods and compounds of the present invention are useful for increasingexpression of genes associated with iron transport and absorption.Additionally, these results suggested that compounds of the presentinvention increase iron absorption and transport in healthy subjects andin subjects with anemia of chronic disease.

Example 21 Enhanced Erythropoiesis in Human Subjects

The effect of prolyl hydroxylase inhibition on erythropoiesis in humansubjects was examined as follows. An oral dose of 20 mg/kg of compound Awas administered either two or three times per week for four weeks tohealthy human volunteers. At various times following compoundadministration, blood was drawn for analysis of EPO, hemoglobin,hematocrit, red blood cell counts, soluble transferrin receptor, andserum ferritin levels.

Reticulocyte Count

As shown in FIG. 20, administration of compound A to human subjectsincreased reticulocyte counts above that of placebo control. Increasedreticulocyte counts occurred in subjects administered compound twice orthree-times weekly. Reticulocyte levels increased to greater thanapproximately 1.7% of red blood cells in treated individuals, comparedto levels of approximately 1.4% in non-treated individuals. Compound Aadministration increased reticulocyte counts in human subjects.Therefore, methods and compounds of the present invention are useful forenhancing erythropoiesis and thereby increasing reticulocyte levels.

Hematocrit

Hematocrit levels were increased in human subjects treated with compoundA. In human subjects administered compound A twice weekly for threeweeks, hematocrit levels were greater than 46% compared to approximately44% in placebo control subjects. Compound A increased hematocrit inhuman subjects. Therefore, compounds and methods of the presentinvention are useful for enhancing erythropoiesis and thereby increasinghematocrit.

Red Blood Cell Count

Administration of compound A increased red blood cell count in humansubjects. As shown in FIG. 21, red blood cell counts were increased inhuman subjects treated with 20 mg/kg compound A, either twice weekly orthree-times per week, compared to non-treated placebo control subjects.These data indicated that methods and compounds of the present inventionare useful for enhancing erythropoiesis and thereby increasing red bloodcell count.

Iron Status—Soluble Transferrin Receptor and Serum Ferritin

Results shown above indicated methods and compounds of the presentinvention are effective at increasing reticulocyte count, red bloodcells, hemoglobin, and hematocrit in human subjects. As shown in FIG.22, administration of compound A to human subjects increased solubletransferrin receptor levels above that observed in non-treated controlsubjects. Increased soluble transferrin levels were observed humansubjects treated twice or three-times weekly. A maximum response of 35%and 31% was observed on day 21 in patients treated 2-times and 3-timesper week, respectively. Mean plasma concentrations of sTfR in placebopatients was unchanged. Additionally, serum ferritin levels decreasedapproximately 46% in human subject treated with compound A, indicativeof increased iron utilization in these subjects. (See FIG. 23.)

Taken together, these data indicated that HIF stabilization usingcompounds and methods of the present invention resulted in increasedmobilization of iron stores, increased transport of iron to bone marrow,and increased utilization of iron for hemoglobin synthesis,erythropoiesis, and red cell production.

Various modifications of the invention, in addition to those shown anddescribed herein, will become apparent to those skilled in the art fromthe foregoing description. Such modifications are intended to fallwithin the scope of the appended claims.

All references cited herein are hereby incorporated herein by referencein their entirety.

What is claimed is:
 1. A method for treating anemia in a subject in needthereof, the method comprising administering to the subject an effectiveamount of a heterocyclic carboxamide compound that stabilizes the alphasubunit of hypoxia-inducible factor (HIFα), thereby increasing thepercent transferrin saturation in the subject, wherein the subject is asubject having a percent transferrin saturation level below 20% prior tosaid administering.
 2. The method of claim 1, wherein the heterocycliccarboxamide compound is a heterocyclic carbonyl glycine compound.
 3. Themethod of claim 1, wherein the subject further has a serum ferritinlevel above 200 ng/ml, above 300 ng/ml, or above 400 ng/ml.
 4. Themethod of claim 1, wherein the subject further has a serum ferritinlevel below 200 ng/ml, below 150 ng/ml, below 100 ng/ml, or below 50ng/ml.
 5. The method of claim 1, wherein the subject further has atransferrin saturation level below 16%, below 15%, or below 10%.
 6. Themethod of claim 1, wherein the method increases the transferrinsaturation level in the subject to a level above 20%, above 30%, above40%, or above 50%.
 7. The method of claim 1, wherein the treating anemiais achieved without administering iron to the subject.
 8. The method ofclaim 1, wherein the anemia is anemia of chronic disease.
 9. The methodof claim 1, wherein the subject is a subject having a chronic diseaseselected from the group consisting of an inflammation, an infection, animmunodeficiency disorder, and a neoplastic disorder.
 10. The method ofclaim 1, wherein the subject is a subject having iron deficiency. 11.The method of claim 10, wherein the iron deficiency is functional irondeficiency.
 12. The method of claim 1, wherein the heterocycliccarboxamide compound inhibits hypoxia-inducible factor prolylhydroxylase enzyme activity.
 13. The method of claim 1, wherein theheterocyclic carboxamide compound is a structural mimetic of2-oxoglutarate.